Compositions and methods including a recombinant human MAB that promotes CNS remyelination

ABSTRACT

Antibodies, and particularly human antibodies, are disclosed that demonstrate activity in the treatment of demyelinating diseases as well as other diseases of the central nervous system that are of viral, bacterial or idiopathic origin, including neural dysfunction caused by spinal cord injury. Neuromodulatory agents are set forth that include and comprise a material selected from the group consisting of an antibody capable of binding structures or cells in the central nervous system, a peptide analog, a hapten, active fragments thereof, agonists thereof, mimics thereof, monomers thereof and combinations thereof. Methods are described for treating demyelinating diseases, and diseases of the central nervous system of humans and domestic animals, using polyclonal IgM antibodies and human monoclonal antibodies sHIgm22(LYM 22), sHIgm46(LYM46) ebvHIgM MSI19D10, CB2bG8, AKJR4, CB2iE12, CB2iE7, MSI19E5 and MSI10E10, active fragments thereof and the like. The invention also extends to the use of human antibodies, fragments, peptide derivatives and like materials, and their use in above referenced therapeutic applications, and to pharmaceutical compositions containing them, that may be administered in desirably low doses to treat conditions involving demyelination and to promote remyelination.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation Application of co-pending U.S.application Ser. No. 13/788,529, filed on Mar. 7, 2013, which is aContinuation Application of Ser. No. 10/557,115, filed on Dec. 13, 2006,now U.S. Pat. No. 8,460,665, which in turn, claims priority fromApplication Serial No. PCT/US2004/015436, filed on May 17, 2004, whichclaims priority from U.S. Provisional Application Ser. No. 60/471,235filed May 16, 2003. Applicants claim the benefits of 35 U.S.C. §120 asto the said Applications, and the entire disclosures of bothapplications are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to the field of neurobiology,and more particularly to the identification of recombinantly producedantibodies that play a role in central nervous system function andtherapy. The invention also relates to therapeutic materials andmethods, including by way of example, pharmaceutical compositions,methods of treatment of diseases associated with neurologicalimpairment, methods of regeneration and restoration of neural function,screening assays and vaccines.

BACKGROUND OF THE INVENTION

Multiple sclerosis (MS) is a chronic, frequently progressive,inflammatory central nervous system (CNS) disease characterizedpathologically by primary demyelination, usually without initial axonalinjury. The etiology and pathogenesis of MS are unknown. Severalimmunological features of MS, and its moderate association with certainmajor histocompatibility complex alleles, has prompted the speculationthat MS is an immune-mediated disease.

An autoimmune hypothesis is supported by the experimental autoimmune(allergic) encephalomyelitis (EAE) model, where injection of certainmyelin components into genetically susceptible animals leads to Tcell-mediated CNS demyelination. However, specific autoantigens andpathogenic myelin-reactive T cells have not been definitively identifiedin the CNS of MS patients, nor is MS associated with other autoimmunediseases. An alternative hypothesis, based upon epidemiological data, isthat an environmental factor, perhaps an unidentified virus,precipitates an inflammatory response in the CNS, which leads to eitherdirect or indirect (“bystander”) myelin destruction, potentially with aninduced autoimmune component. This hypothesis is supported by evidencethat several naturally occurring viral infections, both in humans andanimals, can cause demyelination. One commonly utilized experimentalviral model is induced by Theiler's murine encephalomyelitis virus(TMEV) (Dal Canto, M. C., and Lipton, H. L., Am. J. Path., 88:497-500(1977)).

The limited efficacy of current therapies for MS and other demyelinatingdiseases, has stimulated interest in novel therapies to ameliorate thesediseases. However, due to the apparently complex etiopathogenesis ofthese diseases, potentially involving both environmental and autoimmunefactors, the need still exists for an effective treatment of thesedemyelinating disorders.

rHIgM22 is a recombinant human IgM antibody that binds to matureoligodendrocytes and myelin of both rodents and humans, and promotes thesynthesis of new myelin in in vivo models of demyelination. The standarddose of remyelination-promoting mAbs in prior studies has been 25 mg/kg,administered IP. This dose, if extrapolated to humans, would beimpractical.

Accordingly, a need exists to develop a practical, safe and efficacioustreatment regimen for CNS disorders, particularly those involvingdemyelination and/or remyelination, and it is toward the fulfillment ofthat need that the present invention is directed.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication.

SUMMARY OF THE INVENTION

In accordance with the present invention, human antibodies have beencloned and isolated that demonstrate activity in the promotion,stimulation, regeneration and/or remyelination of neurons in the centralnervous system, and/or in the blocking or reduction of demyelination inthe central nervous system. Specifically, the present invention relatesto methods of stimulating the remyelination of central nervous system(CNS) axons using recombinant autoantibodies, and particularlyrecombinant human autoantibodies, including antibodies of the IgMsubtype and monomers thereof, or mixtures and/or active fragmentsthereof, characterized by their ability to bind to structures and cellsin the central nervous system, particularly including oligodendrocytes.

In a first aspect of the invention, the antibody is a human antibodysuch as that designated rHIgM22, and the composition includes suchantibody in an amount effective to promote remyelination in the centralnervous system. The antibody and the corresponding composition areprepared to deliver doses ranging from about 500 ng to about 600 μg,calculated on a per kg of body weight basis. In a particular embodiment,the doses may be on the order of 500 ng, or may be on the order of 600μg.

In a particular embodiment, the rHIgM22 antibody is administered alonein a pharmaceutically acceptable composition. In another embodiment, therHIgM22 antibody is administered in combination with methylprednisolone.The methylprednisolone may be administered at doses ranging from about 1to 2 mg once or twice a week.

In another embodiment, the administration of the rHIgM22 antibody withmethylprednisolone may be concurrent or it may be sequential.

A second aspect of the invention provides for pharmaceuticalcompositions comprising a therapeutically effective amount of therHIgM22 antibody and a pharmaceutically acceptable carrier. In oneembodiment, the composition comprises a therapeutically effective amountof the rHIgM22 antibody alone. In another embodiment, the compositioncomprises a therapeutically effective amount of the rHIgM22 antibody incombination with a therapeutically effective amount ofmethylprednisolone. In yet another embodiment, the composition of therHIgM22 antibody may be formulated as one composition, and themethylprednisolone may be formulated as a separate composition, and eachmay be delivered to a subject in need of such therapy sequentially orconcurrently. In yet another embodiment, the therapeutically effectiveamount of the rHIgM22 antibody is an amount that promotes remyelinationof neurons. In another embodiment, the therapeutically effective amountof the rHIgM22 antibody is an amount that prevents demyelination. In yetanother embodiment, the therapeutically effective amount of the rHIgM22antibody is an amount that decreases demyelination while also promotingremyelination. When used in combination with methylprednisolone, therHIgM22 antibody may be more effective at preventing demyelination,promoting remyelination or a combination thereof.

A fourth aspect of the invention provides fragments and monomers derivedfrom or related to the recombinant human antibodies of the presentinvention. Thus, the invention particularly extends to fragments, ormonomers derived from or based on sHIgM22 (LYM 22). Such fragments andor monomers possess the same activity as the parent antibody moleculeand may demonstrate the capability to remyelinate neurons or to preventdemyelination of neurons.

A fifth aspect of the invention provides an assay for screening otherantibodies and related binding partners, including haptens and peptideanalogs, that may exhibit a like therapeutic activity. Such activitieswould include the treatment or prevention of neurological injuries ordysfunctions such as multiple sclerosis, ALS, stroke, Parkinsons diseaseand Aizheimers disease.

A sixth aspect of the invention provides methods of treatingdemyelinating diseases in mammals, such as multiple sclerosis in humans,and viral diseases of the central nervous system of humans and domesticanimals, such as post-infectious encephalomyelitis, or prophylacticallyinhibiting the initiation or progression of demyelination in thesedisease states, using the recombinant monoclonal antibodies, or activefragments thereof, of this invention.

This invention further relates to in vitro methods of producing andstimulating the proliferation of glial cells, such as oligodendrocytes,and the use of these glial cells to treat demyelinating diseases.Accordingly, in one embodiment, the demyelinating neurological conditionor disorder for which such antibody therapy would be effective ismultiple sclerosis. In another embodiment, the demyelinatingneurological condition or disorder for which such antibody therapy wouldbe effective is acute or chronic spinal cord injury. Other neurologicalconditions or diseases in which demyelination of nerves or nerve fibersis prominent are also contemplated.

The present invention also extends to the cloning, isolation and use ofrecombinant human autoantibodies that aid in remyelination of neurons orprevent demyelination of neurons, which human autoantibodies areexemplified by rHIgM22. The following terms are used interchangeablythroughout this application: RsHIgM22, sHIgM22, rHIgM22 and LYM 22. Theheavy and light chain variable region sequences of the recombinantrHIgM22 antibody are set forth in FIGS. 5 and 6 and accordingly, theinvention extends to antibodies and corresponding antibody proteins, andsmall molecules such as haptens, that have or correspond at least inpart to the sequences set forth in the noted Figures. These sequences ofthe antibody may be used in part for cloning the human recombinant formof the antibody, or may be used as probes for research or diagnosticpurposes. The invention extends further in that the newly identifiedrecombinant antibodies may be employed for a variety of purposes such asthe promotion of remyelination, regeneration of damaged nerve cells,neuronal protection, neuronal outgrowth and the like.

The invention is also broadly directed to peptides which bind to theautoantibodies described herein, whereby these peptides by virtue oftheir sequence, three-dimensional structure, or conformational changesarising from antibody binding, can be used in and of themselves aspeptide vaccines. In a further aspect of the invention, these peptidesmay have neuromodulatory and/or immunomodulatory properties and mayprovide a method of inducing a neural cell proliferative response and/orneuroprotective, neuroregenerative and/or remyelinating role in mammalsin need of such therapy.

Likewise, the invention includes haptens that may bind to the peptides,the antibodies and/or other relevant substrates and that may possessimmunogenicity, so that they may also function as active components intherapeutic formulations, also including vaccines. In a particularembodiment, one or more haptens may be combined with other of thepeptides of the present invention, in a vaccine formulation.

In yet a further aspect of the invention these peptides can beformulated as pharmaceutical compositions with stabilizers to preventproteolytic degradation, thus extending their half-life to be givenorally, subcutaneously, intravenously, intranasally, intrathecally or asliposome preparations to mammals in need of such therapy.

In a further aspect, the invention extends to a group of molecules thatwill be referred to herein as neuromodulatory agents, and that arenotable in their therapeutic activity in the CNS. Accordingly, theinvention relates to neuromodulatory agents with particulareffectiveness in the CNS, which agents comprise a material selected fromthe group consisting of an antibody of the IgM subtype, a peptideanalog, a hapten, active fragments thereof, monomers thereof, agoniststhereof, mimics thereof, and combinations thereof. Related antibodies ofdifferent subtypes, including those that have undergone a class switch(naturally or as generated by recombinant or synthetic means), are alsocontemplated, wherein the class switch antibodies have characteristicsas neuromodulatory agents useful in the methods of the presentinvention. The neuromodulatory agents have one or more of the followingcharacteristics: they induce remyelination and/or cellular proliferationof glial cells; and/or evoke Ca⁺⁺ signaling with oligodendrocytes;and/or block cell death, e.g. hydrogen-peroxide induced cell death.

The antibodies of the present invention may be used in conjunction withother antibodies that bind to neural tissue, such as polyclonalantibodies that may also induce remyelination, in particular otherpolyclonal IgM antibodies, particularly polyclonal IgM immunoglobulinand preparations thereof, more particularly pooled polyclonal IgMimmunoglobulin, and pooled polyclonal human IgM immunoglobulin.Preferably, the antibody is a recombinantly produced human antibody or arecombinantly produced chimeric antibody capable of remyelination. Inanother particular embodiment, the recombinant antibody is rHIgM22(LYM22), monomers thereof, active fragments thereof, and natural orsynthetic antibodies having the characteristics of rHIgM22. Theinvention provides antibodies comprising a polypeptide having an aminoacid sequence corresponding at least in part to a sequence selected fromFIGS. 5 and 6, and active fragments thereof.

The present invention also relates to a recombinant DNA molecule orcloned gene, or a degenerate variant thereof, which encodes a class ofmolecules that will also be referred to herein as neuromodulatoryagents, and that include and may be selected from the antibodies of theinvention, and particularly antibodies having sequences corresponding atleast in part, to the sequences presented in FIGS. 5 and 6; peptidesthat may correspond at least in part to the antibodies of the presentinvention, that will also be referred to herein as antibody peptides,and for example, peptides having one or more sequences corresponding atleast in part to FIGS. 5 and 6; and small molecules such as haptens;including recombinant DNA molecules or cloned genes having the same orcomplementary sequences.

The present invention also includes proteins derived from orcorresponding to said antibodies, or fragments or derivatives thereof,having the activities noted herein, and that display the amino acidsequences set forth and described above and selected at least in part,from the group consisting of FIGS. 5 and 6.

The present invention likewise extends to haptens that demonstrate thesame activities as the proteins or antibody peptides, and that may beadministered for therapeutic purposes in like fashion, as by formulationin a vaccine. In one embodiment, a vaccine including both peptides andhaptens may be prepared.

In a further embodiment of the invention, the full DNA sequence of therecombinant DNA molecule or cloned gene so determined may be operativelylinked to an expression control sequence which may be introduced into anappropriate host. The invention accordingly extends to unicellular hoststransformed with the cloned gene or recombinant DNA molecule comprisinga DNA sequence encoding the present antibody peptides.

In a particular embodiment, the variable region DNA sequence of anantibody of the present invention may be utilized in generatingsynthetic antibody(ies). In particular, variable region sequence may becombined with its natural or a genetically provided constant regionsequence to provide a synthetic antibody. The present invention providesvectors for generating synthetic antibodies derived from and comprisingthe DNA sequences, particularly variable region sequences, of theantibodies of the present invention.

According to other preferred features of certain preferred embodimentsof the present invention, a recombinant expression system is provided toproduce biologically active animal or particularly human antibodypeptides.

The present invention includes several means for the preparation ofclones of the autoantibodies, peptides, corresponding haptens, or othersmall molecule analogs thereof, including as illustrated herein knownrecombinant techniques, and the invention is accordingly intended tocover such synthetic preparations within its scope. The isolation of thecDNA and amino acid sequences disclosed herein facilitates thereproduction of the present antibodies or their analogs by suchrecombinant techniques, and accordingly, the invention extends toexpression vectors prepared from the disclosed DNA sequences forexpression in host systems by recombinant DNA techniques, and to theresulting transformed hosts.

The invention includes an assay system for screening of potential drugseffective to modulate the neurological activity of target mammalianneural cells by, for example, potentiating the activity of the presentautoantibodies or their analogs. In one instance, the test drug could beadministered to a cellular sample with the ligand that suppresses orinhibits the activity of the autoantibodies, or an extract containingthe suppressed antibodies, to determine its effect upon the bindingactivity of the autoantibodies to any chemical sample (including DNA),or to the test drug, by comparison with a control.

The present invention includes an assay system which may be prepared inthe form of a test kit for the quantitative analysis of the extent ofthe presence of the neuromodulatory agents, or to identify drugs orother agents that may mimic or block their activity. The system or testkit may comprise a labeled component prepared by one of the radioactiveand/or enzymatic techniques discussed herein, coupling a label to theneuromodulatory agents, their agonists and/or antagonists, and one ormore additional immunochemical reagents, at least one of which is a freeor immobilized ligand, capable either of binding with the labeledcomponent, its binding partner, one of the components to be determinedor their binding partner(s).

In a particular and further aspect, the present invention extends to theuse and application of the antibodies of the present invention,particularly autoantibodies, including antibodies of the IgM subtype andmonomers thereof, or mixtures and/or active fragments thereof,characterized by their ability to bind to structures and cells in thecentral nervous system, particularly including oligodendrocytes, inimaging and in vivo diagnostic applications. Thus, the antibodies, byvirtue of their ability to bind to structures and cells in the centralnervous system, particularly including oligodendrocytes, can be utilizedvia immunofluorescent, radioactive and other diagnostically suitabletags as imaging agents or imaging molecules for the characterization ofthe nervous system, including the central nervous system and thediagnosis, monitoring and assessment of nervous disease, particularlyincluding multiple sclerosis. The antibodies may further be utilized asimaging agents or imaging molecules in the diagnosis, monitoring andassessment of stroke, spinal cord injury and various dementias includingAlzheimer's disease.

In a further embodiment, the present invention relates to certaintherapeutic methods which would be based upon the activity of theneuromodulatory agents, their subunits, or active fragments thereof,peptide equivalents thereof, analogs thereof, or upon agents or otherdrugs determined to possess the same activity. A first therapeuticmethod is associated with the prevention of the manifestations ofconditions causally related to or following from the binding activity ofthe antibodies or their subunits, and comprises administering an agentcapable of stimulating the production and/or activity of theneuromodulatory agents, the corresponding autoantibodies, antibodypeptides, active fragments or subunits thereof, either individually orin mixture with each other in an amount effective to prevent or treatthe development of those conditions in the host. For example, drugs orother binding partners to the antibodies or their fragments, or thelike, may be administered to potentiate neuroregenerative and/orneuroprotective activity, or to stimulate remyelination as in thetreatment of multiple sclerosis.

More specifically, the therapeutic method generally referred to hereincould include the method for the treatment of various pathologies orother cellular dysfunctions and derangements by the administration ofpharmaceutical compositions that may comprise effective inhibitors orenhancers of activation of the neuromodulatory agents, or other equallyeffective drugs developed for instance by a drug screening assayprepared and used in accordance with an aspect of the present inventiondiscussed above. For example, drugs or other binding partners to theneuromodulatory agents or like proteins, having sequences correspondingat least in part to the sequences as represented by FIGS. 5 and 6, maybe administered to inhibit or potentiate neuroregeneration,neuroprotection, or remyelination, as in the treatment of Parkinsonsdisease or multiple sclerosis. In particular, the proteins of sHIgM22(LYM22), whose sequence is presented in FIGS. 5 and 6, their antibodies,agonists, antagonists, monomers or active fragments thereof, includingmixtures and combinations thereof, could be prepared in pharmaceuticalformulations including vaccines, for administration in instances whereinneuroregenerative and/or neuroprotective therapy or remyelination isappropriate, such as to treat Alzheimers disease, ALS, Parkinsonsdisease, or spinal cord injury. The present invention includescombinations or of the antibodies provided herein, wherein theantibodies, particularly human antibodies, most particularly selectedfrom sHIgM22 can be prepared in pharmaceutical and therapeuticcompositions or formulations. Combinations or mixtures of various humanantibodies, mouse antibodies, or monomers, fragments, recombinant orsynthetic antibodies derived therefrom or based thereon are alsoprovided by and included in the present invention. The human antibodies(extending to monomers, fragments, recombinant or synthetic antibodiesderived therefrom) are particularly selected from the group of sHIgM22,sHIgM46, MSI19E10, CB2bG8, AKJR4, CB2iE12, CB2iE7, MSI19E5, andMSI10E10. The mouse antibodies (extending to monomers, fragments,recombinant or synthetic antibodies and humanized antibodies derivedtherefrom) are particularly selected from the group of SCH 94.03,SCH79.08, 01, 04, 09, A2B5 and HNK-1. In addition, the inventionprovides further combinations of the antibody(ies) with therapeuticcompounds, drugs or agents useful in any such neuroregenerative and/orneuroprotective therapy or remyelination. For instance, the antibodyformulation or composition of the present invention may be combined withtherapeutic compounds for the treatment of multiple sclerosis, includingbut not limited to beta interferon formulations (Betaseron, etc.) andcoploymer 1 (Copaxone). In addition, the antibodies of the presentinvention may be combined with other agents that may act to inhibitinflammation at the site of injury. One such agent may bemethylprednisolone.

Accordingly, it is a principal object of the present invention toprovide neuromodulatory agents, including the human autoantibodies andcorresponding antibody peptides, haptens, analogs and active fragmentsthereof in purified form that exhibits certain characteristics andactivities associated with the promotion of neuroregenerative and/orneuroprotective activity.

It is a further object of the present invention to provide a method fordetecting the presence, amount and activity of the autoantibodies inmammals in which invasive, spontaneous, or idiopathic pathologicalstates are suspected to be present.

It is a further object of the present invention to provide a method andassociated assay system for screening substances such as drugs, agentsand the like, potentially effective in either mimicking the activity orcombating any adverse effects of the autoantibodies and/or theirfragments, subunits or the like, in mammals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the results of a comparative dose ranging study,with varying concentrations of rHIgM22, placebo, methylprednisolonealone and in combination with rHIgM22.

FIG. 2 is a graph of the mean scores of the test subject groups in thedose ranging study.

FIG. 3 is a graph demonstrating that rHIgM22 combined withmethylprednisolone promotes remyelination and reduces lesion load.

FIG. 4 is a graph of the results of a comparative dose ranging studywith varying concentrations of rHIgM22.

FIG. 5 presents the sHIgM22 heavy chain variable region sequences. Thesequence is aligned according to the numbering system of human V_(H)sequences in the publication: Sequences of Proteins of ImmunologicalInterest, Vol I, Fifth Edition (1991), Kabat E. A., Wu, T. T., Perry, H.M. Gottesman, K. S. and Poeller, C., NIH Publication. The sHIgM22 V_(H)is a member of the V_(H) subgroup III. Underlined amino acids have beenconfirmed by protein sequencing. Amino acid sequence corresponds tosHIgM22 nucleotide sequence. SHIgM22 V_(H) type A and B sequences arerepresented only with nucleotides that differ from theIGHV3-30/3-30-05*01, IGHJ4*02 and IGHD2-21*02 germline sequences. Twoamino acid replacements in the protein sequence of sHIgM22 V_(H) type Bare printed in bold. sHIgM22 antibody heavy chain variable region aminoacid sequence is set out in SEQ ID NO: 7, and the nucleic acid sequenceis in SEQ ID NO: 8. The sequences of both sHIgM22 V_(H) type A and Bmost closely matched the IGHV3-30/3-30-5*01 germline sequence (96%homology). References for germline sequences: IMGT, the internationalImMunoGeneTics database [imgt.cnusc]. (Initiator and coordinator:Marie-Paule Lefranc, Montpellier, France)

FIG. 6 presents the sHIgM22 light chain variable region sequences. Thesequence is aligned according to the numbering system of human V_(H)sequences in the publication: Sequences of Proteins of ImmunologicalInterest, Vol I, Fifth Edition (1991), Kabat E. A., Wu, T. T., Perry, H.M. Gottesman, K. S. and Foeller, C., NIH Publication. Vλ. sHIgM22 is amember of the lambda subgroup I. Underlined amino acids have beenconfirmed by protein sequencing. Amino acid sequence corresponds tosHIgM22 nucleotide sequence. SHIgM22 Vλ type I and II sequences arerepresented only with nucleotides that differ from the IGLV1-51*01 andIGLJ3*01 germline sequences. Two amino acid replacements in the proteinsequence of sHIgM22 Vλ; type II are printed in bold. sHIgM22 antibodylight chain variable region amino acid sequence is set out in SEQ ID NO:9, and the nucleic acid sequence is in SEQ ID NO: 10. The Vλ sequencesfrom SHIgM22 most closely matched the IGLV-51*01 germline sequence (97%homology). The two genes differ from their common ancestor by a singlenucleotide change. References for germline sequences: IMGT, theinternational ImMunoGeneTics database [imgt.cnusc]. (Initiator andcoordinator: Marie-Paule Lefranc, Montpellier, France).

DETAILED DESCRIPTION OF THE INVENTION

Before the present methods and treatment methodology are described, itis to be understood that this invention is not limited to particularmethods, and experimental conditions described, as such methods andconditions may vary. It is also to be understood that the terminologyused herein is for purposes of describing particular embodiments only,and is not intended to be limiting, since the scope of the presentinvention will be limited only in the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein and/or which will become apparent to those persons skilled in theart upon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference.

Definitions

Also as used herein, the terms “rHIgM” and “rsHIgM”, “sHIgM22” and“LYM22” as pertains to the antibodies of the invention, shall beconsidered equivalent herein.

“Subject” includes humans. The terms “human,” “patient” and “subject”are used interchangeably herein.

“Therapeutically effective amount” means the amount of a compound that,when administered to a subject for treating a disease, is sufficient toeffect such treatment for the disease. The “therapeutically effectiveamount” can vary depending on the compound, the disease and its severityand the age, weight, etc., of the subject to be treated.

“Treating” or “treatment” of any disease or disorder refers, in oneembodiment, to ameliorating the disease or disorder (i.e., arresting orreducing the development of the disease or at least one of the clinicalsymptoms thereof). In another embodiment “treating” or “treatment”refers to ameliorating at least one physical parameter, which may not bediscernible by the subject. In yet another embodiment, “treating” or“treatment” refers to modulating the disease or disorder, eitherphysically, (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter), or both.In yet another embodiment, “treating” or “treatment” refers to delayingthe onset of the disease or disorder.

The term “neuromodulatory agent(s)” as used herein singularly throughoutthe present application and claims, is intended to refer to a broadclass of materials that function to promote neurite outgrowth,regeneration and remyelination with particular benefit and effect in theCNS, and therefore includes the antibodies of the IgM sub-type, andparticularly, human antibodies such as those referred to specificallyherein as sHIgM22 (LYM 22), ebvHIgM MSI19D10, sHIgM46 (LYM46), CB2bG8,AKJR4, CB2iE12, CB2iE7 and MSI19E5, peptide analogs, haptens, activefragments thereof, monomers thereof, agonists, mimics and the like,including such materials as may have at least partial sequencesimilarity to the peptide sequences set forth in FIGS. 5 and 6 and inSEQ ID NOS: 7 and 9. Neuromodulatory agent(s) also includes andencompasses combinations or mixtures of more than one of the antibodiesprovided herein, including monomers or active fragments thereof.

Also, the terms “neuromodulatory agent,” “autoantibody,” “antibodypeptide,” “peptide,” “hapten” and any variants not specifically listed,may be used herein interchangeably, to the extent that they may allrefer to and include proteinaceous material including single or multipleproteins, and extends to those proteins having the amino acid sequencedata described herein and presented in FIGS. 5-6 and in SEQ ID NOS: 7and 9, and the profile of activities set forth herein and in the claims.Accordingly, proteins displaying substantially equivalent or alteredactivity are likewise contemplated. These modifications may bedeliberate, for example, such as modifications obtained throughsite-directed mutagenesis, or may be accidental, such as those obtainedthrough mutations in hosts that are producers of the complex or itsnamed subunits. Also, the terms “neuromodulatory agent,” “autoantibody,”“antibody peptide,” “peptide,” “hapten” are intended where appropriate,to include within their scope proteins specifically recited herein aswell as all substantially homologous analogs and allelic variations.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunctional property of immunoglobulin-binding is retained by thepolypeptide. NH₂ refers to the free amino group present at the aminoterminus of a P COOH refers to the free carboxy group present at thecarboxy terminus of a polypeptide. In keeping with standard polypeptidenomenclature, J. Biol. Chem., 243:3552-59 (1969), abbreviations foramino acid residues are shown in the following Table of Correspondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGln glutamic acid W Trp tryptophan R Arg arginine D Asp aspartic acid NAsn aspargine C Cys cysteine

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues. The above Table ispresented to correlate the three-letter and one-letter notations whichmay appear alternately herein.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. A “DNA molecule” refers to the polymeric formof deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in itseither single stranded form, or a double-stranded helix. This termrefers only to the primary and secondary structure of the molecule, anddoes not limit it to any particular tertiary forms. Thus, this termincludes double-stranded DNA found, inter alia, in linear DNA molecules(e.g., restriction fragments), viruses, plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA).

An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined by mapping with nuclease S1), as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequencesin addition to the −10 and −35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

The term “oligonucleotide,” as used herein in referring to probes of thepresent invention, is defined as a molecule comprised of two or moreribonucleotides, preferably more than three. Its exact size will dependupon many factors which, in turn, depend upon the ultimate function anduse of the oligonucleotide.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA polymerase and at a suitable temperature and pH. The primer maybe either single-stranded or double-stranded and must be sufficientlylong to prime the synthesis of the desired extension product in thepresence of the inducing agent. The exact length of the primer willdepend upon many factors, including temperature, source of primer anduse of the method. For example, for diagnostic applications, dependingon the complexity of the target sequence, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides.

The primers herein are selected to be “substantially” complementary todifferent strands of a particular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being complementary to the strand.Alternatively, non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence of the strand to hybridizetherewith and thereby form the template for the synthesis of theextension product.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra. In particular, the heavy chainand light chain variable region sequences of the antibodies of thepresent invention are substantially homologous to a correspondinggermline gene sequence, having at least about 90% homology to acorresponding germline gene sequence.

It should be appreciated that also within the scope of the presentinvention are DNA sequences encoding an antibody of the invention, or apeptide analog, hapten, or active fragment thereof, which code for apeptide that defines in at least a portion thereof, or has the sameamino acid sequence as set forth in FIGS. 5-6 and SEQ ID NOS: 7 and 9,but which are degenerate to the same SEQ ID NOS. By “degenerate to” ismeant that a different three-letter codon is used to specify aparticular amino acid. It is well known in the art that the followingcodons can be used interchangeably to code for each specific amino acid:

Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or L)UUA or UUG or CUU or CUC or CUA or CUG Isoleucine (Ile or I)AUU or AUC or AUA Methionine (Met or M) AUG Valine (Val or V)GUU or GUC of GUA or GUG Serine (Ser or S)UCU or UCC or UCA or UCG or AGU or AGC Proline (Pro or P)CCU or CCC or CCA or CCG Threonine (Thr or T) ACU or ACC or ACA or ACGAlanine (Ala or A) GCU or GCG or GCA or GCG Tyrosine (Tyr or Y)UAU or UAC Histidine (His or H) CAU or CAC Glutamine (Gln or Q)CAA or CAG Asparagine (Asn or N) AAU or AAC Lysine (Lys or K) AAA or AAGAspartic Acid (Asp or D) GAU or GAC Glutamic Acid (Glu or E) GAA or GAGCysteine (Cys or C) UGU or UGC Arginine (Arg or R)CGU or CGC or CGA or CGG or AGA or AGG Glycine (Gly or G)GGU or GGC or GGA or GGG Tryptophan (Trp or W) UGG Termination codonUAA (ochre) or UAG (amber) or UGA (opal)

It should be understood that the codons specified above are for RNAsequences. The corresponding codons for DNA have a T substituted for U.

Mutations can be made in a particular DNA sequence or molecule such thata particular codon is changed to a codon which codes for a differentamino acid. Such a mutation is generally made by making the fewestnucleotide changes possible. A substitution mutation of this sort can bemade to change an amino acid in the resulting protein in anon-conservative manner (i.e., by changing the codon from an amino acidbelonging to a grouping of amino acids having a particular size orcharacteristic to an amino acid belonging to another grouping) or in aconservative manner (i.e., by changing the codon from an amino acidbelonging to a grouping of amino acids having a particular size orcharacteristic to an amino acid belonging to the same grouping). Such aconservative change generally leads to less change in the structure andfunction of the resulting protein. A non-conservative change is morelikely to alter the structure, activity or function of the resultingprotein. The present invention should be considered to include sequencescontaining conservative changes which do not significantly alter theactivity or binding characteristics of the resulting protein.

The following is one example of various groupings of amino acids:

Amino Acids with Nonpolar R Groups

Alanine

Valine

Leucine

Isoleucine

Proline

Phenylalanine

Tryptophan

Methionine

Amino Acids with Uncharged Polar R Groups

Glycine

Serine

Threonine

Cysteine

Tyrosine

Asparagine

Glutamine

Amino Acids with Charged Polar R Groups (Negatively Charged at pH 6.0)

Aspartic acid

Glutamic acid

Basic Amino Acids (Positively Charged at pH 6.0)

Lysine

Arginine

Histidine (at pH 6.0)

Another grouping may be those amino acids with phenyl groups:

Phenylalanine

Tryptophan

Tyrosine

Another grouping may be according to molecular weight (i.e., size of Rgroups):

Glycine 75 Alanine 89 Serine 105 Proline 115 Valine 117 Threonine 119Cysteine 121 Leucine 131 Isoleucine 131 Asparagine 132 Aspartic acid 133Glutamine 146 Lysine 146 Glutamic acid 147 Methionine 149 Histidine (atpH 6.0) 155 Phenylalanine 165 Arginine 174 Tyrosine 181 Tryptophan 204

Particularly preferred substitutions are:

Lys for Arg and vice versa such that a positive charge may bemaintained;

Glu for Asp and vice versa such that a negative charge may bemaintained;

Ser for Thr such that a free —OH can be maintained; and

Gin for Asn such that a free NH₂ can be maintained.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site for disulfide bridges with another Cys. AHis may be introduced as a particularly “catalytic” site (i.e., His canact as an acid or base and is the most common amino acid in biochemicalcatalysis). Pro may be introduced because of its particularly planarstructure, which induces β-turns in the protein's structure.

Two amino acid sequences are “substantially homologous” when at leastabout 70% of the amino acid residues (preferably at least about 80%, andmost preferably at least about 90 or 95%) are identical, or representconservative substitutions. In particular, the heavy chain and lightchain variable region sequences of the antibodies of the presentinvention are substantially homologous to a corresponding germline geneamino acid sequence, having at least about 90%, and preferably at leastabout 95% homology to a corresponding germline gene amino acid sequence.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

As used herein, the term “antibody” is any immunoglobulin, includingantibodies and fragments thereof, that binds a specific epitope. Theterm is intended to encompass polyclonal, monoclonal, and chimericantibodies, the last mentioned described in further detail in U.S. Pat.Nos. 4,816,397 and 4,816,567. Such antibodies include both polyclonaland monoclonal antibodies prepared by known generic techniques, as wellas bi-specific or chimeric antibodies, and antibodies including otherfunctionalities suiting them for additional diagnostic use conjunctivewith their capability of modulating activity, e.g. that stimulates theremyelenation and/or regeneration of CNS axons, or that providesneuroprotection. An “antibody combining site” is that structural portionof an antibody molecule comprised of heavy and light chain variable andhypervariable regions that specifically binds antigen. The phrase“antibody molecule” in its various grammatical forms as used hereincontemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule. Exemplaryantibody molecules are intact immunoglobulin molecules, substantiallyintact immunoglobulin molecules and those portions of an immunoglobulinmolecule that contains the paratope, including those portions know inthe art as Fab, Fab′, F(ab′)₂ and F(v).

Fab and F(ab′)₂ portions of antibody molecules, or antibody fragments,may be prepared by the proteolytic reaction of papain and pepsin,respectively, on substantially intact antibody molecules by methods thatare well-known. See for example, U.S. Pat. No. 4,342,566 toTheofilopolous et al. Fab′ antibody molecule portions are alsowell-known and are produced from F(ab′)₂ portions followed by reductionof the disulfide bonds linking the two heavy chains portions as withmercaptoethanol, and followed by alkylation of the resulting proteinmercaptan with a reagent such as iodoacetamide. An antibody containingintact antibody molecules is preferred herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bi-specific(chimeric) monoclonal antibody.

The general methodology for making monoclonal antibodies by hybridomasis well known. Immortal, antibody-producing cell lines can also becreated by techniques other than fusion, such as direct transformationof B lymphocytes with oncogenic DNA, or transfection with Epstein-Barrvirus. See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980);Hammerling et al., “Monoclonal Antibodies And T-cell Hybridomas” (1981);Kennett et al., “Monoclonal Antibodies” (1980); see also U.S. Pat. Nos.4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917;4,472,500; 4,491,632; 4,493,890.

General Description

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-Ill [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984).

Panels of monoclonal antibodies useful in the present invention methodsor produced against neuromodulatory agent peptides or autoantibodypeptides can be screened for various properties; i.e., isotype, epitope,affinity, etc. Of particular interest are monoclonal antibodies thatexhibit the same activity as the neuromodulatory agents, andparticularly the present autoantibodies. Such monoclonals can be readilyidentified in activity assays such as the Theilers virus, EAE andlysolecithin models presented and illustrated herein. High affinityantibodies are also useful when immunoaffinity purification of native orrecombinant autoantibodies is possible.

Preferably, the antibody used in the diagnostic methods and therapeuticmethods of this invention is an affinity purified polyclonal antibody.More preferably, the antibody is a monoclonal antibody (mAb). Inaddition, it is contemplated for the antibody molecules used herein bein the form of Fab, Fab′, F(ab′)₂ or F(v) portions of whole antibodymolecules.

As suggested earlier, the diagnostic method of the present inventioncomprises examining a cellular sample or medium by means of an assayincluding an effective amount of an antagonist to an antibodypeptide/protein, such as an anti-peptide antibody, preferably anaffinity-purified polyclonal antibody, and more preferably a mAb. Inaddition, it is preferable for the anti-peptide antibody molecules usedherein be in the form of Fab, Fab′, F(ab′)₂ or F(v) portions or wholeantibody molecules. As previously discussed, patients capable ofbenefiting from this method include those suffering from a neurologicalcondition such as multiple sclerosis, Alzheimers disease, Parkinsonsdisease, a viral infection or other like neuropathological derangement,including damage resulting from physical trauma. Methods for isolatingthe peptides and inducing anti-peptide antibodies and for determiningand optimizing the ability of anti-peptide antibodies to assist in theexamination of the target cells are all well-known in the art.

Methods for producing polyclonal anti-polypeptide antibodies arewell-known in the art. See U.S. Pat. No. 4,493,795 to Nestor et al. Amonoclonal antibody, typically containing Fab and/or F(ab)₂ portions ofuseful antibody molecules, can be prepared using the hybridomatechnology described in Antibodies—A Laboratory Manual, Harlow and Lane,eds., Cold Spring Harbor Laboratory, New York (1988), which isincorporated herein by reference. Briefly, to form the hybridoma fromwhich the monoclonal antibody composition is produced, a myeloma orother self-perpetuating cell line is fused with lymphocytes obtainedfrom the spleen of a mammal hyperimmunized with an antibodypeptide-binding portion thereof, or the antibody peptide or fragment, oran origin-specific DNA-binding portion thereof.

Splenocytes are typically fused with myeloma cells using polyethyleneglycol (PEG) 6000. Fused hybrids are selected by their sensitivity toHAT. Hybridomas producing a monoclonal antibody useful in practicingthis invention are identified by their ability to immunoreact in thesame fashion as the present autoantibodies and their ability to inhibitor promote specified activity in target cells and tissues.

A monoclonal antibody useful in practicing the present invention can beproduced by initiating a monoclonal hybridoma culture comprising anutrient medium containing a hybridoma that secretes antibody moleculesof the appropriate antigen specificity. The culture is maintained underconditions and for a time period sufficient for the hybridoma to secretethe antibody molecules into the medium. The antibody-containing mediumis then collected. The antibody molecules can then be further isolatedby well-known techniques.

Media useful for the preparation of these compositions are bothwell-known in the art and commercially available and include syntheticculture media, inbred mice and the like. An exemplary synthetic mediumis Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol.8:396 (1959)) supplemented with 4.5 gm/l glucose, 20 mm glutamine, and20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

Methods for producing monoclonal anti-peptide antibodies are alsowell-known in the art. See Niman et al., Proc. Natl. Acad. Sci. USA,80:4949-4953 (1983). Typically, the present antibody peptides, or apeptide analog or fragment, is used either alone or conjugated to animmunogenic carrier, as the immunogen in the before described procedurefor producing anti-peptide monoclonal antibodies. The hybridomas arescreened for the ability to produce an antibody that immunoreacts withthe antibody peptide analog and thereby reacts similarly to theantibodies of the present invention.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays, hemagglutination assays),complement fixation assays, immunofluorescence assays, protein A assays,and immunoelectrophoresis assays, etc. In one embodiment, antibodybinding is detected by detecting a label on the primary antibody. Inanother embodiment, the primary antibody is detected by detectingbinding of a secondary antibody or reagent to the primary antibody. In afurther embodiment, the secondary antibody is labeled. Many means areknown in the art for detecting binding in an immunoassay and are withinthe scope of the present invention.

Antibodies can be labeled for detection in vitro, e.g., with labels suchas enzymes, fluorophores, chromophores, radioisotopes, dyes, colloidalgold, latex particles, and chemiluminescent agents. Alternatively, theantibodies can be labeled for detection in vivo, e.g., withradioisotopes (preferably technetium or iodine); magnetic resonanceshift reagents (such as gadolinium and manganese); or radio-opaquereagents.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others. A number of fluorescent materials are known and canbe utilized as labels. These include, for example, fluorescein,rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. Aparticular detecting material is anti-rabbit antibody prepared in goatsand conjugated with fluorescein through an isothiocyanate. Thepolypeptide can also be labeled with a radioactive element or with anenzyme. The radioactive label can be detected by any of the currentlyavailable counting procedures. The preferred isotope may be selectedfrom ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I,and ¹⁸⁶Re.

Enzyme labels are likewise useful, and can be detected by any of thepresently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques. Theenzyme is conjugated to the selected particle by reaction with bridgingmolecules such as carbodiimides, diisocyanates, glutaraldehyde and thelike. Many enzymes which can be used in these procedures are known andcan be utilized. The preferred are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090;3,850,752; and 4,016,043 are referred to by way of example for theirdisclosure of alternate labeling material and methods.

Polyclonal immunoglobulin preparations have been shown to exert abeneficial clinical effect in various clinical situations that arecharacterized or accompanied by a dysfunction or dysregulation of theimmune system. Immunoglobulin is also used to prevent or treat someillnesses that can occur when an individual does not produce enough ofits own immunity to prevent these illnesses. Nearly all immunoglobulinpreparations in use today are comprised of highly purified IgG, derivedfrom large pools of human plasma by fractionation. These preparationsare commonly administered intravenously (WIG), although intramuscularadministration (IGIM) and oral administration is also used.

Commonly used IgG preparations include Gamimune (5% and 10%) (BayerCorporation), Gammagard (Baxter Healthcare Corporation), Polygam(American Red Cross), Sandoglobin (Sandoz Pharmaceuticals), Venoglobin(Alpha Therapeutic) and Intraglobin (Biotest Pharma GmbH). Anintramuscular immunoglobulin (IGIM), BayGam, is available from BayerCorporation. WIG preparations in clinical use contain predominantly IgG,smaller amounts of IgA, and yet smaller amounts of IgM, IgE and IgD, andgenerally comprise 95% or greater IgG, 2-5% IgA and trace amounts ofIgM.

Pentaglobin (Biotest Pharma GmbH) is an IgM-enriched polyvalentimmunoglobulin preparation and each ml of solution comprises: IgM 6 mg;IgA 6 mg; IgG 38 mg and glucose monohydrate for injection 27.5 mg; or12% IgM, generally 10-15% IgM. Immunoglobulin preparations which havebeen further enriched for IgM can be readily generated and have beenreported as effective in animal models for treatment or alleviation ofcertain conditions. Riebert et al. report the use of IgM enriched humanintravenous immunoglobulin in a rat model of acute inflammation,particularly use of Pentaglobin and a laboratory preparation of IVIgM(35 g/l IgM, 12 g/l IgA, 3 g/l IgG). (Riebert, R. et al (1999) Blood93(3):942-951). Hurez et al report use of an intravenous IgM preparationof greater than 90% in experimental autoimmune ueveitis (EAU) (Hurez, V.et al (1997) Blood 90(10):4004-4013). IgM antibody immunoglobulinpreparations of at least 20% by weight IgM are described in U.S. Pat.Nos. 5,256,771, 5,510,465 and 5,612,033, incorporated herein byreference in their entity. Intravenously administrable polyclonalimmunoglobulin preparations containing at least 50% by weight of IgM interms of the total content of immunoglobulin are described by Moller etal in U.S. Pat. No. 5,190,752, incorporated herein by reference in itsentirety.

Immunoglobulin preparations are generated by methods and processesgenerally well known to those of skill in the art. Immunoglobulins areprepared from blood of healthy volunteers, where the number of blooddonors is at least about 5 or 10; preferably at least about 100; morepreferably at least about 1,000; still more preferably at least about10,000. In one common method, human plasma derived from pools ofthousands of donors is fractionated by cold ethanol fractionation (theCohn process or Cohn-Oncley process) (Cohn, et al (1946) J.Am.Chem.Soc.68:459-475; Oncley, et al (1949) J. Am. Chem. Soc. 71:541-550) followedby enzymatic treatment at low pH, fractionation and chromatography. Coldethanol fractionation may also be followed by ultrafiltration and ionexchange chromatography. Further steps are incorporated to renderimmunoglobulin preparations safe from viral transmission, including butnot limited to enzymatic modification, chemical modification, treatmentwith beta-propiolactone, treatment at low pH, treatment at high heat andtreatment with solvent/detergent. Treatment with an organicsolvent/detergent (S/D) mixture eliminates viral transmission byenveloped viruses (HIV, hepatitis B, hepatitis C) (Gao, F. et al (1993)Vox Sang 64(4):204-9; U.S. Pat. Nos. 4,481,189 and 4,540,573,incorporated herein by reference). Particular processes and methods forpreparation of IgM enriched immunoglobulin solutions are described inU.S. Pat. No. 4,318,902 and U.S. Pat. No. 6,136,132, which areincorporated herein by reference in their entirety.

Polyclonal IgM-enriched immunoglobulin preparations contemplated hereinand suitable for use in the methods of the present invention can be madeby any of the well-known methods used for preparing immunoglobulinpreparations. Suitable immunoglobulin preparations can also be obtainedcommercially. The immunoglobulin preparation can be a humanimmunoglobulin preparation. Suitable immunoglobulin preparations includeat least about 10% IgM, at least about 15% IgM, at least about 20% IgM,at least about 25% IgM, at least about 30% IgM, at least about 40% IgM,at least about 50% IgM, at least about 60% IgM, at least about 70% IgM,at least about 80% IgM, at least about 90% IgM and at least about 95%IgM. Polyclonal IgM immunoglobulin preparations suitable for use in thepresent invention include greater 10% IgM, greater than 20% IgM, andgreater than 50% IgM. Polyclonal IgM immunoglobulin preparationssuitable for use in the present invention include an amount of IgM whichis greater than the amount if IgG and greater than the amount of IgA.

Preparations of fragments of IgM enriched immunoglobulins, particularlyhuman immunoglobulins can also be used in accordance with the presentinvention. Fragments of immunoglobulins refer to portions of intactimmunoglobulins such as Fc, Fab, Fab′, F(ab)′₂ and single chainimmunoglobulins or monomers.

The IgM-enriched immunoglobulin preparation in preferably provided in apharmaceutically acceptable carrier, vehicle or diluent and isadministered intravenously, intramuscularly or orally. IgMimmunoglobulin is administered in doses and amounts similar to theadministration recognized and utilized by the skilled artisan for theadministration of clinically adopted immunoglobulins, including WIG orIGIM or Pentaglobin, or as instructed or advised clinically or by themanufacturer. In accordance with a central aspect of the invention, therecombinant IgM preparations are administered in doses as determined inmice, of from about 500 ng to about 600 μg. By adjusting these amountsfor adaptation to humans, taking into account both the size of thesubject and differences in surface to volume, the approximate rangewould be from about 1.25 to about 2.5 μg/kg body weight. Administrationcan be conducted in a single dose or in multiple separated or divideddoses daily or over the course of days or months. Suitable dosagesinclude 1.25 μg/kg body weight, 1.3 μg/kg body weight, 1.4 μg/kg bodyweight, 1.5 μg/kg body weight, 1.6 μg/kg body weight, 1.7 μg/kg bodyweight, 1.8 μg/kg body weight, 1.9 μg/kg body weight, 2.0 μg/kg bodyweight, 2.1 μg/kg body weight, 2.2 μg/kg body weight, 2.3 μg/kg bodyweight, 2.4 μg/kg body weight, and 2.5 μg/kg body weight. The polyclonalIgM immunoglobulin preparations may be administered alone or incombination with other treatments, including but not limited to othercompounds or agents for treatment or alleviation of the condition. Inthe instance of treatment or alleviation of a demyelinating disease,multiple sclerosis in particular, the IgM immunoglobulin may beadministered with anti-inflammatories, steroids, Betaseron, Copaxone,etc.

Accordingly, in one aspect of the diagnostic application of the presentinvention, a method is disclosed for detecting the presence or activityof a neuromodulatory agent, the neuromodulatory agent comprising amaterial selected from the group consisting of an antibody, a peptideanalog, a hapten, monomers thereof, active fragments thereof, agoniststhereof, mimics thereof, and combinations thereof, said neuromodulatoryagent having one or more of the following characteristics: inducingremyelination; binding to neural tissue; promoting Ca⁺⁺ signaling witholigodendrocytes; and, optionally, promoting cellular proliferation ofglial cells; wherein said neuromodulatory agent is measured by:

A) contacting a biological sample from a mammal in which the presence oractivity of said neuromodulatory agent is suspected with a bindingpartner of said neuromodulatory agent under conditions that allowbinding of said neuromodulatory agent to said binding partner to occur;and

B) detecting whether binding has occurred between said neuromodulatoryagent from said sample and the binding partner;

wherein the detection of binding indicates that presence or activity ofthe neuromodulatory agent in the sample.

In a variant aspect, the invention extends to a method for detecting thepresence and activity of a polypeptide ligand associated with a giveninvasive stimulus in mammals comprising detecting the presence oractivity of the neuromodulatory agent as set forth above, wheredetection of the presence or activity of the neuromodulatory agentindicates the presence and activity of a polypeptide ligand associatedwith a given invasive stimulus in mammals. In a particular aspect, theinvasive stimulus is an infection, and may be selected from viralinfection, protozoan infection, bacterial infection, tumorous mammaliancells, and toxins.

In a further aspect, the invention extends to a method for detecting thebinding sites for a neuromodulatory agent, said neuromodulatory agentcomprising a material selected from the group consisting of an antibody,including antibodies of the IgM subtype and monomers thereof, a peptideanalog, a hapten, active fragments thereof, agonists thereof, mimicsthereof, and combinations thereof, said neuromodulatory agent having oneor more of the following characteristics: inducing remyelination;binding to neural tissue; promoting Ca⁺⁺ signaling witholigodendrocytes; and, optionally, promoting cellular proliferation ofglial cells; said method comprising:

A. placing a labeled neuromodulatory agent sample in contact with abiological sample from a mammal in which binding sites for saidneuromodulatory agent are suspected;

B. examining said biological sample in binding studies for the presenceof said labeled neuromodulatory agent;

wherein the presence of said labeled neuromodulatory agent indicates abinding site for a neuromodulatory agent.

Yet further, the invention includes a method of testing the ability of adrug or other entity to modulate the activity of a neuromodulatoryagent, said agent comprising a material selected from the groupconsisting of an antibody, including antibodies of the IgM subtype, apeptide analog, a hapten, monomers thereof, active fragments thereof,agonists thereof, mimics thereof, and combinations thereof, which methodcomprises:

A. culturing a colony of test cells which has a receptor for theneuromodulatory agent in a growth medium containing the neuromodulatoryagent;

B. adding the drug under test; and

C. measuring the reactivity of said neuromodulatory agent with thereceptor on said colony of test cells;

wherein said neuromodulatory agent has one or more of the followingcharacteristics:

-   -   a) inducing remyelination;    -   b) binding to neural tissue, particularly oligodendrocytes;    -   c) promoting Ca⁺⁺ signaling with oligodendrocytes; and    -   d) promoting cellular proliferation of glial cells.

Correspondingly, the invention covers an assay method for screeningdrugs and other agents for ability to modulate the production or mimicthe activities of a neuromodulatory agent, said neuromodulatory agentcomprising a material selected from the group consisting of an antibody,a peptide analog, a hapten, monomers thereof, active fragments thereof,agonists thereof, mimics thereof, and combinations thereof, said methodcomprising:

A. culturing an observable cellular test colony inoculated with a drugor agent;

B. harvesting a supernatant from said cellular test colony; and

C. examining said supernatant for the presence of said neuromodulatoryagent wherein an increase or a decrease in a level of saidneuromodulatory agent indicates the ability of a drug to modulate theactivity of said neuromodulatory agent, said neuromodulatory agenthaving one or more of the following characteristics:

-   -   i) inducing remyelination;    -   ii) binding to neural tissue, particularly oligodendrocytes;    -   iii) promoting Ca⁺⁺ signaling with oligodendrocytes; and    -   iv) promoting cellular proliferation of glial cells.

Lastly, a test kit is contemplated for the demonstration of aneuromodulatory agent in a eukaryotic cellular sample, saidneuromodulatory agent comprising a material selected from the groupconsisting of an antibody, including antibodies of the IgM subtype andmonomers thereof, a peptide analog, a hapten, active fragments thereof,agonists thereof, mimics thereof, and combinations thereof, which kitcomprises:

A. a predetermined amount of a detectably labeled specific bindingpartner of a neuromodulatory agent, said neuromodulatory agent havingone or more of the following characteristics: inducing remyelination;binding to neural tissue; promoting Ca⁺⁺ signaling witholigodendrocytes; and promoting cellular proliferation of glial cells;

B. other reagents; and

C. directions for use of said kit.

A variant test kit is disclosed for demonstrating the presence of aneuromodulatory agent in a eukaryotic cellular sample, said agentcomprising a material selected from the group consisting of an antibody,a peptide analog, a hapten, monomers thereof, active fragments thereof,agonists thereof, mimics thereof, and combinations thereof. The kitcomprises:

A. a predetermined amount of a neuromodulatory agent, saidneuromodulatory agent having one or more of the followingcharacteristics: inducing remyelination; binding to neural tissue;promoting Ca⁺⁺ signaling with oligodendrocytes; and promoting cellularproliferation of glial cells;

B. a predetermined amount of a specific binding partner of saidneuromodulatory agent;

C. other reagents; and

D. directions for use of said kit;

wherein either said neuromodulatory agent or said specific bindingpartner are detectably labeled. Both of the above kits may utilize alabeled immunochemically reactive component selected from the groupconsisting of polyclonal antibodies to the neuromodulatory agent,monoclonal antibodies to the neuromodulatory agent, fragments thereof,and mixtures thereof.

The present invention extends to the use and application of theantibodies of the present invention, particularly autoantibodies,including antibodies of the IgM subtype and monomers thereof, ormixtures and/or active fragments thereof, characterized by their abilityto bind to structures and cells in the central nervous system,particularly including oligodendrocytes, in imaging and in vivodiagnostic applications. Thus, the antibodies, by virtue of theirability to bind to structures and cells in the central nervous system,particularly including oligodendrocytes, can be utilized viaimmunofluorescent, radioactive and other diagnostically suitable tags asimaging agents or imaging molecules for the characterization of thenervous system, including the central nervous system and the diagnosis,monitoring and assessment of nervous disease, particularly includingmultiple sclerosis. The antibodies may further be utilized as imagingagents or imaging molecules in the diagnosis, monitoring and assessmentof stroke, spinal cord injury and various dementias includingAlzheimer's disease. The appropriate and suitable immunofluorescent,radioactive, or other tagging molecules or agents for coupling orattachment to the antibodies for use in in vivo imaging will be wellknown to and within the skill of the skilled artisan.

The present invention also relates to methods of treating demyelinatingdiseases in mammals, such as multiple sclerosis in humans, and viraldiseases of the central nervous system of humans and domestic animals,such as post-infectious encephalomyelitis, using the recombinant humanantibody described herein as rHIgM22 as well as SCH 94.03, SCH 79.08,01, 04, A2B5 and HNK-1 monoclonal antibodies, and the humanautoantibodies ebvHIgM MSI19D10, sHIgM46, analogs thereof includinghaptens, active fragments thereof, or a natural or syntheticautoantibody having the characteristics thereof. Methods of prophylactictreatment using these mAb, active fragments thereof, or other natural orsynthetic autoantibodies having the same characteristics, to inhibit theinitiation or progression demyelinating diseases are also encompassed bythis invention.

Oligodendrocytes (OLs), the myelin-forming cells of the central nervoussystem (CNS), originate as neuroectodermal cells of the subventricularzones, and then migrate and mature to produce myelin. The sequentialdevelopment of OLs is identified by well-characterized differentiationstage-specific markers. Proliferative and migratory bipolar precursors,designated oligodendrocyte/type-3 astrocyte (O-2A) progenitors, areidentified by monoclonal antibodies (mAbs) anti-GD₃ and A2B5 [Eisenbarthet al., Proc. Natl. Acad. Sci. USA, 76 (1979), 4913-4917]. The nextdevelopmental stage, characterized by multipolar, postmigratory, andproliferative cells, is recognized by mAb 04 [Gard et al., Neuron, 5(1990), 615-625; Sommer et al., Dev. Biol., 83 (1981), 311-327]. Furtherdevelopment is defined by the cell surface expression ofgalactocerebroside, recognized by mAb 01 [Schachner, J. Neurochem., 39(1982), 1-8; Sommer et al., supra], and by the expression of2′,3′-cyclic nucleotide 3′-phosphohydrolase. The most mature cellsexpress terminal differentiation markers such as myelin basic proteinand proteolipid protein.

The mAbs (A2B5, O1, and O4) used to characterize the stages of OLdevelopment were made by immunizing BALB/c mice with chicken embryoretina cells or homogenate of bovine corpus callosum [Eisenbarth et al.,supra; Sommer et al., supra]. A2B5 recognizes not only O-2A progenitorsbut also neurons and reacts with cell surface ganglioside GQ1c [Kasai etal., Brain Res., 277 (1983), 155-158] and other gangliosides [Fredman etal., Arch. Biochem. Biophys., 233 (1984), 661-666]. 04 reacts withsulfatide, seminolipid and cholesterol [Bansal et al., J. Neurosci.Res., 24 (1989), 548-557], whereas O1 reacts with galactocerebroside,monogalactosyl-diglyceride and psychosine [Bansal et al., supra]. ThesemAbs belong to the IgM immunoglobulin (Ig) subclass and recognizecytoplasmic structures as well as the surface antigens of OLs[Eisenbarth et al., supra; Sommer et al., supra]. Mouse mAb HNK-1(anti-Leu-7), made by immunizing BALB/c mice with the membranesuspension of HSB-2 T lymphoblastoid cells, was first reported as amarker for natural killer cells [Abo et al., J. Immunol., 127 (1981),1024-1029]. Later, HNK-1 was shown to share antigenic determinants withthe nervous system [Schuller-Petrovic et al., Nature, 306 (1983),179-181]. The carbohydrate epitope on myelin-associated glycoprotein,found in both central and peripheral myelin sheaths, was shown to be aprincipal antigen of nervous tissue the reacted with FINK-1 [McGarry etal., Nature, 306 (1983), 376-378]. However, other glycoproteins innervous tissue react with this mAb, some of which are important inembryogenesis, differentiation, and myelination [Keilhauer et al.,Nature, 316 (1985), 728-730; Kruse et al., Nature, 311 (1984), 153-155;Kruse et al., Nature, 316 (1985), 146-148; McGarry et al., J.Neuroimmunol., 10 (1985), 101-114]. Of interest, HNK-1 also reacts withcytoplasmic structures and belongs to the IgM Ig subclass.

A monoclonal antibody, disclosed and claimed by certain of the inventorsof the present application in application U.S. Ser. No. 08/236,520,incorporated herein by reference in its entirety, which antibody isdesignated SCH94.03, was found to promote CNS remyelination in miceinfected chronically with Theiler's murine encephalomyelitis virus(TMEV) [Miller et al., J. Neurosci., 14 (1994), 6230-6238]. SCH94.03belongs to the IgM(x) Ig subclass and recognizes an unknown surfaceantigen on OLs, but cytoplasmic antigens in all cells (Asakura et al.,Molecular Brain Research, in press). The polyreactivity of SCH94.03 byELISA, and the unmutated Ig variable region germline sequences indicatedthat SCH94.03 is a natural autoantibody [Miller et al., J. Neurosci., 14(1994), 6230-6238]. A close study of SCH94.03, and comparison thereofwith well-known OL-reactive mAbs A2B5, O1, O4, and FINK-1 raised thepossibility that these are natural autoantibodies. A subsequent analysisof the Ig variable region cDNA sequences and the polyreactivity of thesemAbs by ELISA confirmed that this is a generic group of naturalautoantibodies having similar utilities.

The antigen reactivity of the monoclonal antibody, IgM monoclonalantibody referred to herein as SCH 94.03 (also referred to herein asSCH94.32) and SCH 79.08 (both prepared from a mammal immunized withspinal cord homogenate from a normal mammal (i.e., uninfected with anydemyelinating disease)), have been characterized and described in theaforesaid Application U.S. Ser. No. 08/236,520, filed Apr. 29, 1994,whose teachings are incorporated herein by reference, using severalbiochemical and molecular assays, including immunohistochemistry,immunocytochemistry, Western blotting, solid-phase enzyme-linkedimmunosorbant assays (ELISA), and Ig variable region sequencing. Thehybridomas producing monoclonal antibody SCH 94.03 and SCH 79.08 havebeen deposited on Apr. 28, 1994, and Feb. 27, 1996, respectively, underthe terms of the Budapest Treaty, with the American Type CultureCollection (ATCC) and have been given ATCC Accession Nos. CRL 11627 andHB12057, respectively. All restrictions upon the availability of thedeposit material will be irrevocably removed upon granting of a patent.

Natural or physiologic autoantibodies are present normally in serum, arecharacterized by being reactive or capable of binding to selfstructures, antigens or cells. They are often polyreactive, arefrequently of the IgM subtype, and are encoded by unmutated germlinegenes or are substantially homologous to germline genes with few orseveral sequence differences. By sequencing immunoglobulin (Ig) cDNAs ofthe oligodendrocyte-reactive O1, O4, A2B5, and HNK-1 IgM×monoclonalantibodies and comparing these with published germline sequences, it wasdetermined that these were natural autoantibodies. O1 V_(H) wasidentical with unrearranged V_(H) segment transcript A1 and A4, O4 V_(H)had three and HNK-1 V_(H) had six nucleotide differences from V_(H)101in the V_(H) coding region. The D segment of O1 was derived fromgermline SP2 gene family, J_(H)4, whereas O1 J_(H) was encoded bygermline J_(H)I with one silent nucleotide change. O1 and 04 lightchains were identical with myeloma MOPC21 except for one silentnucleotide change. HNK-1 V_(x) was identical with germline V_(x)41except for two silent nucleotide changes. O1 J_(x), O4J_(x) and HNKJ_(x) were encoded by unmutated germline J_(x)2. In contrast, A2B5 V_(H)showed seven nucleotide differences from germline V1, whereas nogermline sequence encoding A2B5 V_(x) was identified. O1 and O4, but notA2B5 were polyreactive against multiple antigens by direct ELISA.Therefore, O1, O4 and HNK-1 Igs are encoded by germline genes, and havethe genotype and phenotype of natural autoantibodies.

Treatment of Demyelinating Diseases

The results of the experiments described herein have practicalapplications to multiple sclerosis (MS), EAE, and other related centralnervous system demyelinating disorders. Rare examples of spontaneousCNS-type remyelination (“shadow plaques”) are found in MS and occasionalperipheral nervous system (PNS)-type remyelination is found indemyelinated spinal cord plaques near the root entry zone.Oligodendrocytes are infrequent at the center of the chronic plaques inMS but they appear to proliferate at the periphery of plaques, wherethey are associated with abortive remyelination. The process ofremyelination may correlate with the spontaneous remission andimprovements observed clinically in MS. These clinical observationsindicate that new myelin formation is possible in MS. The remyelinationthat has been stimulated in mice with TMEV-induced demyelination byusing a mAb holds promise for therapeutic applications in multiplesclerosis.

Of importance clinically is the question of whether morphologicregeneration of thin myelin sheaths contributes to functional recovery.Computer simulations indicate that new myelin formation even byinappropriately thin sheaths improves impulse conduction. Since the axonmembrane of normally myelinated fibers is highly differentiated, it isnecessary for sodium channels to be present at high density at the nodeof Ranvier to propagate salutatory conduction. Experimental evidencesuggests that newly formed nodes do develop the required high sodiumchannel density as demonstrated by saxitoxin binding. Data to datesuggest that remyelination even by inappropriately thin myelin improvesconduction in a previously demyelinated axon. Therefore, any strategy topromote this morphologic phenomenon has the potential of producingfunctional recovery.

The data presented herein demonstrates, for the first time, thatadministration of a recombinant human monoclonal antibody to a mammal iscapable of stimulating remyelination of central nervous system axons invivo. Specifically, treatment of chronically infected TMEV-infected micewith as little as 500 ng of rHIgM22 resulted in a significant increasein the total area of CNS myelination compared to mice treated with acontrol mAb.

The use of human antibodies avoids the potential for human immuneresponse against the therapeutic antibody. Therapeutic antibodiesderived from non-human animals have been shown to generate an immuneresponse, which can be significant and detrimental to the individual.Accordingly, polyclonal human IgM and polyclonal human IgG have beentested in two models of in vivo spinal cord demyelination; a chronicviral infection model, and an acute toxicity model. In both modelspolyclonal human IgM treated animals had a significantly higher densityof newly myelinated axons than animals treated with polyclonal humanIgG. A panel of human monoclonal IgM antibodies have also beenidentified, based on their reactivity with surface antigens specific tothe central nervous system. These human antibodies promote significantlymore central nervous system remyelination than polyclonal human IgG whengiven to mammals with demyelinating disease. The human monoclonalantibodies are antigenically polyreactive and recognize determinants onthe surface of oligodendrocytes and specific populations of neurons. Thelight and heavy chain variable regions of several human antibodies thatpromote remyelination have been sequenced. In particular, theseantibodies can induce calcium fluxes in glial cells (oligodendrocytesand astrocytes) in culture, suggestive of direct binding and signalingthrough glial cells. These human antibodies bind to human white matterand may be effective in promoting remyelination in humans. The benefitsof a recombinant monoclonal antibody for use as a therapeutic agentare 1) the antibody can be grown free of possible host infection and, 2)the antibody can be genetically altered in vitro to change itseffectiveness.

Thus, as a result of the experiments described herein, the method of thepresent invention can be used to treat mammals, including humans anddomestic animals, afflicted with demyelinating disorders, and tostimulate remyelination and regeneration of the CNS axons, as well as tooffer neuroprotection. As described herein, an effective amount of themonoclonal antibody or a peptide fragment, hapten, or equivalent, can beadministered by conventional routes of administration, and particularlyby, intravenous (iv) or intraperitoneal (ip) injection. As describedherein, therapeutic compositions and vaccines are contemplated and maybe prepared and administered. An effective amount of the antibody canvary depending on the size of the mammal being treated, the severity ofthe disease, the route of administration, and the course of treatment.For example, each dose of antibody administered can range fromapproximately 1.25 to about 2.5 μg/kg, as an exemplary, non limitingrange in accordance with the present invention. The dose of antibodywill also depend on the route of administration. The course of treatmentincludes the frequency of administration of the antibody (e.g., daily,weekly, or bi-weekly) and the duration of the treatment (e.g., fourweeks to four months). Thus, for example, a larger amount of mAb can begiven daily for four to five weeks, as opposed to a smaller amount ofmAb given for four months.

The effectiveness of the amount of the monoclonal antibody beingadministered can be assessed using any number of clinical criteria, forexample, as described in the Examples herein, including overallappearance of the mammal, the activity of the mammal and the extent ofparalysis of the mammal. The effectiveness of the amount of monoclonalantibody necessary to induce remyelination in humans can also beassessed in a double blinded controlled trial. Patients with fixedneurological deficits from demyelinating disease can be treated withmonoclonal antibody or controls. Improvement in isometric musclestrength as detected by quantitative biomechanics muscle testing couldbe used as the primary therapeutic end-point.

In addition to in vivo methods of promoting remyelination, ex vivomethods of stimulating remyelination in CNS axons are also encompassedby the present invention. For example, the monoclonal antibody may beused in vitro to stimulate the proliferation and/or differentiation ofglial cells, such as oligodendrocytes. These exogenous glial cells canthen be introduced into the CNS of mammals using known techniques.Remyelination of CNS axons would be increased by increasing the numberof endogenous glial cells present (glial cells, such as oligodendrocytesplay a critical role in the production of myelin).

In vitro methods of producing glial cells, or stimulating theproliferation of glial cells from mixed culture (e.g., rat optic nervecell, or rat brain cell cultures) are also encompassed by thisinvention. For example, cells obtained from rat optic nerve, or ratbrain, containing glial cells, are cultured as a mixed culture underconditions sufficient to promote growth of the cells. An effectiveamount of mAb capable of promoting remyelination of CNS axons, such asrHIgM22 or sHIgM46, or SCH94.03 or a combination thereof, is then addedto the mixed culture of cells and maintained under conditions sufficientfor growth and proliferation of cells. The mAb stimulates theproliferation of glial cells cultured in the presence of the mAb isincreased, relative to the proliferation of glial cells grown in theabsence of the mAb.

As stated above, the antibodies for use in the methods of the presentinvention can be, and are preferably, administered as medicaments, i.e.,pharmaceutical compositions. An effective amount of the polyclonal IgMantibody can thus be combined with, or diluted with, an appropriatepharmaceutically acceptable carrier, diluent or vehicle, such as aphysiological buffer or saline solution. An effective amount of themonoclonal antibody can thus be combined with, or diluted with, anappropriate pharmaceutically acceptable carrier, diluent or vehicle,such as a physiological buffer, or saline solution. An effective amountof a combination of one or more monoclonal antibody may be similarlycombined with or diluted with an appropriate pharmaceutically acceptablecarrier, diluent or vehicle. In the instance where a vaccine is to beprepared, the monoclonal antibody or equivalent active of the inventionmay be prepared with a pharmaceutically effective and suitable carrieror adjuvant, and the protocol for administration may proceed inaccordance with standard procedures for immunization known to theskilled practitioner.

The pharmaceutical compositions used in the methods of this inventionfor administration to animals and humans comprise the polyclonal IgMantibodies or monoclonal antibodies in combination with a pharmaceuticalcarrier or excipient. In a preferred embodiment, the pharmaceuticalcomposition may contain more than one, preferably two, monoclonalautoantibodies of the present invention. Thus, pharmaceuticalcompositions comprising, for example, an effective amount in combinationof sHIgM22 and sHIgM46 are contemplated herein. Such compositions areadvantageous in that the presence of more than one monoclonalautoantibody will potentiate the activity of others in the sametherapeutic composition or method.

The medicament can be in the form of tablets (including lozenges andgranules), dragees, capsules, pills, ampoules or suppositoriescomprising the compound of the invention.

Advantageously, the compositions are formulated as dosage units, eachunit being adapted to supply a fixed dose of active ingredients.Tablets, coated tablets, capsules, ampoules and suppositories areexamples of preferred dosage forms according to the invention. It isonly necessary that the active ingredient constitute an effectiveamount, i.e., such that a suitable effective dosage will be consistentwith the dosage form employed in single or multiple unit doses. Theexact individual dosages, as well as daily dosages, will, of course, bedetermined according to standard medical principles under the directionof a physician or veterinarian.

The monoclonal antibodies can also be administered as suspensions,solutions and emulsions of the active compound in aqueous or non-aqueousdiluents, syrups, granulates or powders.

Diluents that can be used in pharmaceutical compositions (e.g.,granulates) containing the active compound adapted to be formed intotablets, dragees, capsules and pills include the following: (a) fillersand extenders, e.g., starch, sugars, mannitol and silicic acid; (b)binding agents, e.g., carboxymethyl cellulose and other cellulosederivatives, alginates, gelatine and polyvinyl pyrrolidone; (c)moisturizing agents, e.g., glycerol; (d) disintegrating agents, e.g.,agar-agar, calcium carbonate and sodium bicarbonate; (e) agents forretarding dissolution, e.g., paraffin; (f) resorption accelerators,e.g., quaternary ammonium compounds; (g) surface active agents, e.g.,cetyl alcohol, glycerol monostearate; (g) adsorptive carriers, e.g.,kaolin and bentonite; (i) lubricants, e.g., talc, calcium and magnesiumstearate and solid polyethylene glycols.

The tablets, dragees, capsules and pills comprising the active compoundcan have the customary coatings, envelopes and protective matrices,which may contain opacifiers. They can be so constituted that theyrelease the active ingredient only or preferably in a particular part ofthe intestinal tract, possibly over a period of time. The coatings,envelopes and protective matrices may be made, for example, frompolymeric substances or waxes.

The diluents to be used in pharmaceutical compositions adapted to beformed into suppositories can, for example, be the usual water-solublediluents, such as polyethylene glycols and fats (e.g., cocoa oil andhigh esters, [e.g., C₁₄-alcohol with C₁₆-fatty acid]) or mixtures ofthese diluents.

The pharmaceutical compositions which are solutions and emulsions can,for example, contain the customary diluents (with, of course, theabove-mentioned exclusion of solvents having a molecular weight below200, except in the presence of a surface-active agent), such assolvents, dissolving agents and emulsifiers. Specific non-limitingexamples of such diluents are water, ethyl alcohol, isopropyl alcohol,ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (forexample, ground nut oil, glycerol, tetrahydrofurfuryl alcohol,polyethylene glycols and fatty acid esters of sorbitol or mixturesthereof.

For parental administration, solutions and suspensions should besterile, e.g., water or arachis oil contained in ampoules and, ifappropriate, blood-isotonic.

The pharmaceutical compositions which are suspensions can contain theusual diluents, such as liquid diluents, e.g., water, ethyl alcohol,propylene glycol, surface active agents (e.g., ethoxylated isostearylalcohols, polyoxyethylene sorbitols and sorbitan esters),microcrystalline cellulose, aluminum methahydroxide, bentonite,agar-agar and tragacanth, or mixtures thereof.

The pharmaceutical compositions can also contain coloring agents andpreservatives, as well as perfumes and flavoring additions (e.g.,peppermint oil and eucalyptus oil), and sweetening agents, (e.g.,saccharin and aspartame).

The pharmaceutical compositions will generally contain from 0.5 to 90%of the active ingredient by weight of the total composition.

In addition to the monoclonal antibodies, the pharmaceuticalcompositions and medicaments can also contain other pharmaceuticallyactive compounds, e.g. steroids, anti-inflammatory agents or the like.

Any diluent in the medicaments of the present invention may be any ofthose mentioned above in relation to the pharmaceutical compositions.Such medicaments may include solvents of molecular weight less than 200as the sole diluent.

It is envisaged that the polyclonal IgM antibodies and monoclonalantibodies will be administered perorally, parenterally (for example,intramuscularly, intraperitoneally, subcutaneously, transdermally orintravenously), rectally or locally, preferably orally or parenterally,especially perlingually, or intravenously.

The administered dosage rate will be a function of the nature and bodyweight of the human or animal subject to be treated, the individualreaction of this subject to the treatment, type of formulation in whichthe active ingredient is administered, the mode in which theadministration is carried out and the point in the progress of thedisease or interval at which it is to be administered. Thus, it may insome case suffice to use less than a minimum dosage rate, while othercases an upper limit must be exceeded to achieve the desired results.Where larger amounts are administered, it may be advisable to dividethese into several individual administrations over the course of theday.

According to the invention, the component or components of a therapeuticcomposition of the invention may be introduced parenterally,intrathecally, transmucosally, e.g., orally, nasally, pulmonarally, orrectally, or transdermally. Preferably, administration is parenteral,e.g., via intravenous injection, and also including, but is not limitedto, intra-arterial, intramuscular, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial administration. Oralor pulmonary delivery may be preferred to activate mucosal immunity;since the bacteria responsible for the conditions under treatmentgenerally colonize the nasopharyngeal and pulmonary mucosa, mucosaladministration may be particularly effective as a treatment. The term“unit dose” when used in reference to a therapeutic composition of thepresent invention refers to physically discrete units suitable asunitary dosage for humans, each unit containing a predetermined quantityof active material calculated to produce the desired therapeutic effectin association with the required diluent; i.e., carrier, or vehicle.

In another embodiment, the active compound can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid).

In yet another embodiment, the therapeutic compound can be delivered ina controlled release system. For example, the polypeptide may beadministered using intravenous infusion, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. In oneembodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit.Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment,polymeric materials can be used (see Medical Applications of ControlledRelease, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974);Controlled Drug Bioavailability, Drug Product Design and Performance,Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J.Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al.,Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989);Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment,a controlled release system can be placed in proximity of thetherapeutic target, i.e., the brain, thus requiring only a fraction ofthe systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 115-138 (1984)). Preferably, acontrolled release device is introduced into a subject in proximity ofthe site of inappropriate immune activation or a tumor. Other controlledrelease systems are discussed in the review by Langer (Science249:1527-1533 (1990)).

A subject in whom administration of an active component as set forthabove is an effective therapeutic regimen for a condition or pathologyassociated with the central nervous system, including in certaininstances, bacterial infection is preferably a human, but can be anyanimal. Thus, as can be readily appreciated by one of ordinary skill inthe art, the methods and pharmaceutical compositions of the presentinvention are particularly suited to administration to any animal,particularly a mammal, and including, but by no means limited to,domestic animals, such as feline or canine subjects, farm animals, suchas but not limited to bovine, equine, caprine, ovine, and porcinesubjects, wild animals (whether in the wild or in a zoological garden),research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs,cats, etc., i.e., for veterinary medical use.

In the therapeutic methods and compositions of the invention, atherapeutically effective dosage of the active component is provided. Atherapeutically effective dosage can be determined by the ordinaryskilled medical worker based on patient characteristics (age, weight,sex, condition, complications, other diseases, etc.), as is well knownin the art. Furthermore, as further routine studies are conducted, morespecific information will emerge regarding appropriate dosage levels fortreatment of various conditions in various patients, and the ordinaryskilled worker, considering the therapeutic context, age and generalhealth of the recipient, is able to ascertain proper dosing. Generally,for intravenous injection or infusion, dosage may be lower than forintraperitoneal, intramuscular, or other route of administration. Thedosing schedule may vary, depending on the circulation half-life, andthe formulation used. The compositions are administered in a mannercompatible with the dosage formulation in the therapeutically effectiveamount. Precise amounts of active ingredient required to be administereddepend on the judgment of the practitioner and are peculiar to eachindividual. Suitable regimes for initial administration and boostershots are also variable, but are typified by an initial administrationfollowed by repeated doses at one or more hour intervals by a subsequentinjection or other administration. Alternatively, continuous intravenousinfusion sufficient to maintain concentrations of ten nanomolar to tenmicromolar in the blood are contemplated.

Administration with Other Compounds

For treatment of a demyelinating condition, for instance multiplesclerosis, one may administer the present active component inconjunction with one or more pharmaceutical compositions used fortreating multiple sclerosis, including but not limited to (1)anti-inflammatory agents, such as steroids; (2) Betaseron; (3) Copaxone;or 94) polyclonal IgM, or 4) methylprednisolone. Administration may besimultaneous (for example, administration of a mixture of the presentactive component and an antibiotic), or may be in seriatim.

Accordingly, in specific embodiment, the therapeutic compositions mayfurther include an effective amount of the active component, and one ormore of the following active ingredients: an antibiotic, a steroid, etc.

Also contemplated herein is pulmonary delivery of the presentneuromodulatory agent or agents, which may be associated with ananti-inflammatory. Reports of preparation of proteins for pulmonarydelivery are found in the art [Adjei et al. Pharmaceutical Research,7:565-569 (1990); Adjei et al., International Journal of Pharmaceutics,63:135-144 (1990) (leuprolide acetate); Braquet et al., Journal ofCardiovascular Pharmacology, 13(suppl. 5):143-146 (1989) (endothelin-1);Hubbard et al., Annals of Internal Medicine, Vol. III, pp. 206-212(1989) (al-antitrypsin); Smith et al., J. Clin. Invest. 84:1145-1146(1989) (α-1-proteinase); Oswein et al., “Aerosolization of Proteins”,Proceedings of Symposium on Respiratory Drug Delivery II, Keystone,Colo., March, (1990) (recombinant human growth hormone); Debs et al., J.Immunol. 140:3482-3488 (1988) (interferon-γ and tumor necrosis factoralpha); Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colonystimulating factor)]. A method and composition for pulmonary delivery ofdrugs is described in U.S. Pat. No. 5,451,569, issued Sep. 19, 1995 toWong et al.

All such devices require the use of formulations suitable for thedispensing of adhesin inhibitory agent (or derivative). Typically, eachformulation is specific to the type of device employed and may involvethe use of an appropriate propellant material, in addition to the usualdiluents, adjuvant and/or carriers useful in therapy. Also, the use ofliposomes, microcapsules or microspheres, inclusion complexes, or othertypes of carriers is contemplated. Chemically modified adhesininhibitory agent may also be prepared in different formulationsdepending on the type of chemical modification or the type of deviceemployed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise neuromodulatory agent (orderivative) dissolved in water at a concentration of about 0.1 to 25 mgof biologically active agent per ml of solution. The formulation mayalso include a buffer and a simple sugar (e.g., for neuromodulatoryagent stabilization and regulation of osmotic pressure). The nebulizerformulation may also contain a surfactant, to reduce or prevent surfaceinduced aggregation of the neuromodulatory agent caused by atomizationof the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the neuromodulatory agent(or derivative) suspended in a propellant with the aid of a surfactant.The propellant may be any conventional material employed for thispurpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, ahydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol, and1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactantsinclude sorbitan trioleate and soya lecithin. Oleic acid may also beuseful as a surfactant.

The liquid aerosol formulations contain neuromodulatory agent and adispersing agent in a physiologically acceptable diluent. The dry powderaerosol formulations of the present invention consist of a finelydivided solid form of neuromodulatory agent and a dispersing agent. Witheither the liquid or dry powder aerosol formulation, the formulationmust be aerosolized. That is, it must be broken down into liquid orsolid particles in order to ensure that the aerosolized dose actuallyreaches the mucous membranes of the nasal passages or the lung. The term“aerosol particle” is used herein to describe the liquid or solidparticle suitable for nasal or pulmonary administration, i.e., that willreach the mucous membranes. Other considerations, such as constructionof the delivery device, additional components in the formulation, andparticle characteristics are important. These aspects of pulmonaryadministration of a drug are well known in the art, and manipulation offormulations, aerosolization means and construction of a delivery devicerequire at most routine experimentation by one of ordinary skill in theart. In a particular embodiment, the mass median dynamic diameter willbe 5 micrometers or less in order to ensure that the drug particlesreach the lung alveoli [Wearley, L. L., Crit. Rev. in Ther. Drug CarrierSystems 8:333 (1991)].

The neuromodulatory agents of the invention may also be prepared foradministration in the form of vaccines, which may comprise as theactive, the herein recited autoantibodies, peptide analogs, or haptens,or possibly combinations thereof. Thus, the preparation of vaccines mayproceed in accordance with known procedures, and monovalent as well aspolyvalent vaccines are contemplated. Also, DNA sub unit vaccines, basedupon the DNA molecules of the invention, may be prepared. All vaccinesmay be administered in accordance with standard practices of thephysician or clinician, and such parameters are considered to be withinthe scope of the present invention.

Vectors containing e.g. a DNA-based vaccine in accordance with theinvention can be introduced into the desired host by methods known inthe art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem.267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut etal., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

The vaccine can be administered via any parenteral route, including butnot limited to intramuscular, intraperitoneal, intravenous, and thelike. Preferably, since the desired result of vaccination is toelucidate an immune response to the antigen, and thereby to thepathogenic organism, administration directly, or by targeting or choiceof a viral vector, indirectly, to lymphoid tissues, e.g., lymph nodes orspleen, is desirable. Since immune cells are continually replicating,they are ideal target for retroviral vector-based nucleic acid vaccines,since retroviruses require replicating cells.

Passive immunity can be conferred to an animal subject suspected ofsuffering an autoimmune-mediated demyelinating disease, e.g. multiplesclerosis, by administering antiserum, polyclonal antibodies, or aneutralizing monoclonal antibody to the patient. Preferably, theantibodies administered for passive immune therapy are autologousantibodies. For example, if the subject is a human, preferably theantibodies are of human origin or have been “humanized,” in order tominimize the possibility of an immune response against the antibodies.The active or passive vaccines of the invention, or the administrationof an adhesin, can be used to protect an animal subject from infectionof a Gram positive bacteria, preferably streptococcus, and morepreferably, pneumococcus.

Further, the present invention contemplates treatment by gene therapy,where the appropriate neuromodulatory agent is correspondinglyintroduced to target cells for treatment, to cause or increaseexpression of the corresponding agent. Thus, in one embodiment, the DNAor a gene encoding the neuromodulatory agent, autoantibody, antibodypeptide, etc., or a protein or polypeptide domain fragment thereof isintroduced in vivo, ex vivo, or in vitro using a viral vector or throughdirect introduction of DNA. Expression in targeted tissues can beeffected by targeting the transgenic vector to specific cells, such aswith a viral vector or a receptor ligand, or by using a tissue-specificpromoter, or both.

Viral vectors commonly used for in vivo or ex vivo targeting and therapyprocedures are DNA-based vectors and retroviral vectors. Methods forconstructing and using viral vectors are known in the art [see, e.g.,Miller and Rosman, BioTechniques 7:980-990 (1992)].

DNA viral vectors include an attenuated or defective DNA virus, such asbut not limited to herpes simplex virus (HSV), papillomavirus, EpsteinBarr virus (EBV), adenovirus, adeno-associated virus (AAV), and thelike. Defective viruses, which entirely or almost entirely lack viralgenes, are preferred. Defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Thus, adipose tissue can bespecifically targeted. Examples of particular vectors include, but arenot limited to, a defective herpes virus 1 (HSV1) vector [Kaplitt etal., Molec. Cell. Neurosci. 2:320-330 (1991)], defective herpes virusvector lacking a glyco-protein L gene [Patent Publication RD 371005 A],or other defective herpes virus vectors [International PatentPublication No. WO 94/21807, published Sep. 29, 1994; InternationalPatent Publication No. WO 92/05263, published Apr. 2, 1994]; anattenuated adenovirus vector, such as the vector described byStratford-Perricaudet et al. [J. Clin. Invest. 90:626-630 (1992); seealso La Salle et al., Science 259:988-990 (1993)]; and a defectiveadeno-associated virus vector [Samulski et al., J. Virol. 61:3096-3101(1987); Samulski et al., J. Virol. 63:3822-3828 (1989); Lebkowski etal., Mol. Cell. Biol. 8:3988-3996 (1988)].

Preferably, for in vivo administration, an appropriate immunosuppressivetreatment is employed in conjunction with the viral vector, e.g.,adenovirus vector, to avoid immuno-deactivation of the viral vector andtransfected cells. For example, immunosuppressive cytokines, such asinterleukin-12 (IL-12), interferon-γ (IFN-γ), or anti-CD4 antibody, canbe administered to block humoral or cellular immune responses to theviral vectors [see, e.g., Wilson, Nature Medicine (1995)]. In addition,it is advantageous to employ a viral vector that is engineered toexpress a minimal number of antigens.

In another embodiment the DNA or gene can be introduced in a retroviralvector, e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346;Mann et al., 1983, Cell 33:153; Temin et al., U.S. Pat. No. 4,650,764;Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., 1988, J. Virol.62:1120; Temin et al., U.S. Pat. No. 5,124,263; International PatentPublication No. WO 95/07358, published Mar. 16, 1995, by Dougherty etal.; and Kuo et al., 1993, Blood 82:845. Retroviral vectors can beconstructed to function as infections particles or to undergo a singleround of transfection. In the former case, the virus is modified toretain all of its genes except for those responsible for oncogenictransformation properties, and to express the heterologous gene.Non-infectious viral vectors are prepared to destroy the viral packagingsignal, but retain the structural genes required to package theco-introduced virus engineered to contain the heterologous gene and thepackaging signals. Thus, the viral particles that are produced are notcapable of producing additional virus.

Targeted gene delivery is described in International Patent PublicationWO 95/28494, published October 1995.

Alternatively, the vector can be introduced in vivo by lipofection. Forthe past decade, there has been increasing use of liposomes forencapsulation and transfection of nucleic acids in vitro. Syntheticcationic lipids designed to limit the difficulties and dangersencountered with liposome mediated transfection can be used to prepareliposomes for in vivo transfection of a gene encoding a marker [Feigner,et. al., Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417 (1987); see Mackey,et al., Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031 (1988); Ulmer et al.,Science 259:1745-1748 (1993)]. The use of cationic lipids may promoteencapsulation of negatively charged nucleic acids, and also promotefusion with negatively charged cell membranes [Feigner and Ringold,Science 337:387-388 (1989)]. The use of lipofection to introduceexogenous genes into the specific organs in vivo has certain practicaladvantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. It is clear that directing transfectionto particular cell types would be particularly advantageous in a tissuewith cellular heterogeneity, such as pancreas, liver, kidney, and thebrain. Lipids may be chemically coupled to other molecules for thepurpose of targeting [see Mackey, et. al., supra]. Targeted peptides,e.g., hormones or neurotransmitters, and proteins such as antibodies, ornon-peptide molecules could be coupled to liposomes chemically.

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Naked DNA vectors for gene therapy can be introduced into thedesired host cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter [see, e.g., Wu et al., J. Biol. Chem. 267:963-967(1992); Wu and Wu, J. Biol. Chem. 263:14621-14624 (1988); Hartmut etal., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990;Williams et al., Proc. Natl. Acad. Sci. USA 88:2726-2730 (1991)].Receptor-mediated DNA delivery approaches can also be used [Curiel etal., Hum. Gene Ther. 3:147-154 (1992); Wu and Wu, J. Biol. Chem.262:4429-4432 (1987)].

In a preferred embodiment of the present invention, a gene therapyvector as described above employs a transcription control sequence thatcomprises the DNA consensus sequence recognized by e.g. an autoantibodyof the invention, i.e., an antibody binding site, operably associatedwith a therapeutic heterologous gene inserted in the vector. That is, aspecific expression vector of the invention can be used in gene therapy.

The present invention will be better understood from a consideration ofthe following non-limiting examples, which describe the preparation ofmaterials, compounds and compositions and the development and practiceof methods illustrative of the present invention. It will be apparent tothose skilled in the art that many modifications, both of materials andmethods, may be practiced without departing from the purpose and intentof this disclosure. The following examples are presented in order tomore fully illustrate the preferred embodiments of the invention andserve also in fulfillment of applicants' duty to present the best modeknown for the practice of the invention, and should in no way beconstrued as limiting the broad scope thereof.

EXAMPLES Example 1: Dose Ranging Study Using Recombinant sHIgM22 (Study#1) Mice

4-6 weeks old female &HA mice from the Jackson Laboratories wereinjected intracerebrally with 2×10⁵ plaque-forming units of Daniel'sstrain TMEV in 10 μl. Infected animals received a single 500 μlintraperitoneal (IP) injection of rHIgM22 at various concentrations, orPBS 6 months after TMEV infection. One group received an additional 500μg dose of rHIgM22 after 5 weeks. Two groups of mice received 2 mg ofmethylprednisolone once each week. Ten mice were used for each group.Animals were killed for quantitative analysis of remyelination in thespinal cords after 5 or 10 week of treatment.

To determine the minimum effective dose, a dose-ranging study ofRsHIgM22 was performed in mice chronically infected with TMEV. After 5wk of treatment, spinal cords were removed and graded for demyelinationand remyelination. The results are directly presented in FIG. 1, and acomparison of mean values of the scores categorized as to subjectsreceiving the same dosing, is presented in FIG. 2.

From a review of the results, all doses of RsHIgM22 down to 0.25 mg/kgresulted in significantly greater area of remyelination than salinecontrols (p<0.001 by ANOVA). This demonstrates the ability of theantibodies of the invention to offer therapeutically relevant effects atreasonable dosing.

In addition, animals treated with RsHIgM22 and 2 mg ofmethylprednisolone per week presented with less demyelination (p<0.001)by ANOVA) as well as increased remyelination. This is significantbecause steroids are a primary method of treatment for many patientswith acute exacerbations, who would be potential recipients ofremyelinating human mAbs. Moreover, these results suggest that theaspect of the invention pertaining to the preparation and administrationof a composition comprising a steroid such as methylprednisolone, andthe antibodies of the invention, or alternatively the formulation andadmininstration of a composition comprising the steroid and the antibodyfor separate but conjoint or sequential administration, can yield theadvantageous results demonstrated herein.

These results demonstrate that RsHIgM22 can act at concentrations in therange of those required for classic growth factors.

Example 2: Testing the Effectiveness of rHIgM22 Alone at Lower Doses orin Combination with Methylprednisolone (Study #2)

Materials and Methods

Mice

4-6 weeks old female SJL/J mice from the Jackson Laboratories wereinjected intracerebrally with 2×10⁵ plaque-forming units of Daniel'sstrain TMEV in 10 μl. Infected animals received a single 5000intraperitoneal (IP) injection of rHIgM22 at various concentrations, orPBS 6 months after TMEV infection. One group received an additional 500μg dose of rHIgM22 after 5 weeks. Two groups of mice received 1 mg ofmethylprednisolone twice each week. Ten mice were used for each group.Animals were killed for quantitative analysis of remyelination in thespinal cords after 5 or 10 week of treatment.

Antibody Isolation and Sequencing

IgM antibody was isolated from the serum (designated sHIgM22) of apatient with Waldenstrom's macroglobulinemia and was sequenced asdescribed (Ciric, B. et al (2000), Blood 97: 321-323).

Cloning and Testing of rHIgM22

Vector Construction

1) Light Chain Fragment

The light chain variable region (VL) was amplified from a TA-cloned VLfragment derived from an antibody mRNA isolated from peripheral bloodleukocytes of patient who was the source of sHIgM22. A 5′-Nhe I site,5′-UTR and artificial leader sequence from a human IgM sequence in thehuman genome database (Tsujimoto, Y. et al. (1984), Nucleic Acids Res.12: 8407-8414) were added to this sequence. The constant region ofλ-chain (Cλ) was amplified from human blood cDNA with additional 3′-AvrII site that is found in human Cλ. All polymerase chain reactions (PCRs)were performed by the standard methods by using the following primersdesigned for one identified form of the predominant IgM species(designated light chain: II) found in sHIgM22 (Ciric, B. et al (2000),Blood 97: 321-323):

ctagctagccgaatttcgggacaatcttcatcatgacctgctcccctctcctcctcacccttctcattcactgcacagggtcctgggcccagtctgtgttgacgcagccg (SEQ ID NO: 6) (SEQ ID NO: 1)3′-primer of VL: gggcagccttgggctgacctaggacggtcagc 5′-primer of Cλ:(SEQ ID NO: 2) ctagctagcgtcctaggtcagcccaaggctgccccc 3′-primer of Cλ:(SEQ ID NO: 3) atagtttagcggccgcacctatgaacattctgtagg

2) Heavy Chain Fragment

Human IgM genomic sequence was isolated and cloned by adding two uniquesites on both ends of VH (5′: Rsr II, 3′: PacI). VH was amplified fromTA-coned sHIgM22 VH region with additional artificial 5′-UTR, and leadersequence from the human genome database (11) by using the primers of:

5′-primer of VH: (SEQ ID NO: 4)gactcggaccgcccagccactggaagtcgccggtgtttccattcggtgatcatcactgaacacagaggactcaccatggagtttggctgagctgggtatcctcgttgctcattaagaggtgtccagtgtcaggtgcagctggtggagtct gg 3′-primer of VH:(SEQ ID NO: 5) ccttaattaagacctggagaggccattcttacctgaggagacggtgacca gggttcVH of the human IgM was replaced by this amplified VH fragment ofsHIgM11.

3) Dehydroxy Folic Acid Reductase Gene (dhfr) Fragment

The dhfr fragment was amplified from the vector pFR2000 (Simonsen, C. etal. (1983), PNAS USA 80: 2495-2499) by standard PCR reaction and wasligated into pCICA. The HIgM2211 light chain combined with dhfr wasproduced by EagI digestion of pCICA, and this fragment was ligated intothe Eag I site in sHIgM22 VH vector (pDM 22BII). The gene order of thispDM 22BII vector was 1) heavy chain (genomic), 2) light chain (cDNA),and 3) dhfr. The heavy chain and light chain genes were oriented in thesame direction but that of dhfr was opposite.

Electroporation, Methotrexate (MTX) Amplification, ELISA, and IgMPurification

pDM 22BII was transfected into 2×10 7 of F3B6 cells by electroporation;10 g of pMD22BII linealized by Bg1 II was mixed with 2×10 7 of F3B6cells, resuspended in 8001 of ice-cold serum-free RPMI on ice for 10min, pulsed once at 960 F/200V with Bio-Rad Gene Pulser (Bio-Rad,Hercules, Calif.), returned to ice for 30 min, transferred to a 25-cm 2flask, fed with 5 ml of RPMI with 10% fetal calf serum, and incubated in5% CO2 at 37 ûC. After incubation for 48 h, the medium was replaced byfresh medium containing 0.2 μM of MTX, and incubation continued untilsurvival colonies appeared. These colonies were transferred to a 24-wellplate and were cultured in 1 M MTX. ELISA was performed for these clonesafter confluent growth as described below, and positive clones werechosen for further manipulation. NUNC Maxisorp™ plates were coated with20 μg/ml goat anti-human IgA+IgM+IgG (H+L) (ICN Pharmaceuticals, Inc.,Colta Mesa, Calif.). Following a blocking step with 1% bovine serumalbumin (BSA; Sigma-Aldrich Co., St. Louis, Mo.), supernatants fromsHIgM22BII expressing F3B6 cells were plated in duplicate at 1:500,1:1000, and 1:2000 dilutions. Purified Human IgM (Organon Teknika Corp.West Chester, Pa.) was used as the standard control. After incubationand washes with phosphate buffered saline (PBS)/1% Tween 20, monoclonalanti-human IgM conjugated to alkaline phosphatase (Sigma-Aldrich Co.)was applied at a 1:5000 dilution in 1% BSA. Positive reactions weredetected by using p-Nitrophenyl Phosphate (Sigma-Aldrich Co.), andadsorbance was read on a Bio-Rad microplate reader at 405 nm. MTXconcentration was increased geometrically starting from 0.2 μM and endedat 200 i M over a course of 2 months. The highest antibody-producingclone was determined by ELISA, and the culture media of this clone wasCollected. IgM antibody of recombinant sHIgM22BII was isolated from thismedia by the methods of PEG6000 (Fluka, Buchs, Switzerland)precipitation and dialysis against H2O, followed by size fractionationover a Superose 6 column.

Evaluation of Spinal Cord for Demyelination/Remyelination

Regions of demyelination and remyelination of the spinal cord werevisualized using 4% para-phenylenediamine stained plastic embedded crosssections. To obtain a representative sampling of the entire spinal cord,1 μm thick cross sections were cut from every third serial 1 mm block.This generated 10 to 12 cross sections that represent samples from thecervical, thoracic, lumbar, and sacral spinal cord. For grading eachspinal cord section was divided visually into four quadrants, then eachquadrant was scored for the presence or absence of a demyelinated orremyelinated lesion. All slides were coded and read blind. Data was notassembled into treatment groups until all slides were graded. Lesionswere judged to be remyelinated when the entire lesion was essentiallyrepaired. Partially remyelinated lesions were scored as negative. Levelsof remyelination were calculated as follows: (the number of quadrantswith remyelination/the number of total demyelinated quadrants)×100. Thecategorical data were evaluated using a Chi-square statistical analysis.

Immunohistochemistry

Adult SJL mouse cerebellar slice sections were prepared as describedpreviously (Warrington, A. et al. (1992), J. Neurosci Res. 33:338-353).Briefly, a fresh cerebellum was embedded in 3% low melting temperatureagarose, mounted on a #2 Whatman filter, and cut into 300 μm saggitalslices. Slices were then transferred to 48-well tissue culture plates.Following a 2- to 3-h incubation with 5% BSA inN-(2-hydroxyethyl)piperazine-N-ethanesulfonic acid (HEPES) bufferedEarle™s balanced salt solution (E/H, pH 7.4), the slices were labeledwith primary antibodies at 10 μg/ml in 1% BSA in E/H, for at least 3 hwith gentle rocking at 4° C. After being washed with E/H, the sliceswere stained with appropriate secondary antibodies, washed, and thenfixed briefly with 4% paraformaldehyde in PBS. Slides were mounted inMOWIOL (Aldrich Chemical, Milwaukee, Wis.) containing 2.5%1,4-diazobicyclo-[2.2.2]-octane (DABCO, Sigma, St. Louis, Mo.) andviewed with an epifluorescence microscope. Mixed primary glial cells andpurified oligodendrocytes were prepared from Sprague-Dawley rat neonatesas previously described (Asakura, K. et al. (1997), J. Neurochem 68:2281-2290). Cells were plated on poly-D-lysine-coated orpoly-L-ornithine glass coverslips. Live surface staining was performedat 4 ûC for 15 min on unfixed cells after blocking with E/H containing3% normal goat serum. Bound primary antibodies (Abs) were detected withfluorescence-conjugated secondary Abs. Slides were mounted and viewed asdescribed above.

Purification of rHIgM22

rHIgM22-transfected F3B6 cells were grown in roller bottles in RPMI/10%heat-inactivated FBS/penicillin/streptomycin/glutamine supplemented with10 μM methotrexate (MTX). Conditioned medium was concentrated bytangential flow filtration (TFF) to 0.2 mg/ml of protein. Concentratedconditioned medium was dialyzed overnight against dH₂O to precipitateIgM antibodies. Pellet was resuspended in 50 mM Tris (Trizma base), pH8.0, 150 mM NaCl, 0.5% w/v betaine. The resuspended pellet was loadedonto a Sephacryl S-300 (26/60) column and eluted with 20 mM Tris, pH8.0, 200 mM NaCl, 0.5% betaine. The elution profile consisted of ˜2major peaks: HMW (˜80%) and LMW (˜20%). Pentameric IgM was found in theHMW peak. Pooled GF fractions were concentrated to ˜0.5 mg/ml (A₂₈₀) anddialyzed against 20 mM sodium phosphate, 200 mM NaCl, pH8.0 (finalformulation buffer).

Results

In this experiment, further studies were conducted to determine whethereffective dosing could be achieved with greater reductions inconcentration of the antibody as either the sole active ingredient, orin combination with a steroid such as methylprednisolone. The resultsestablish that even greater reductions and consequent advantages indosing are possible (FIGS. 3 and 4). RHIgM22 was effective at preventingdemyelination at a dose of 600 μg when combined with methylprednisolone(FIG. 3). Furthermore, rHIgM22 was significantly effective atremyelination at doses as low as 500 ng (FIG. 4) when used alone, andalso demonstrated even greater remyelination capabilities whenadministered at a dose of 600 μg when combined with methylprednisolone.

From the foregoing description, various modifications and changes in thecompositions and methods of this invention will occur to those skilledin the art. All such modifications coming within the scope of theappended claims are intended to be included therein.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

What is claimed is:
 1. A therapeutically effective pharmaceuticalcomposition comprising: an antibody capable of stimulating remyelinationcomprising the heavy chain variable regions CDR1, CDR2 and CDR3sequences as set out in FIG. 5 and SEQ ID NO:7 and the light chainvariable region CDR1, CDR2 and CDR3 sequences as set out in FIG. 6 andSEQ ID NO:9; the antibody selected from the group consisting of: a humanmonoclonal antibody mAb sHIgM22 (LYM 22), active fragments thereof, orrecombinant antibodies derived therefrom; a therapeutic compound, drug,or agent useful in neuroregenerative therapy or remyelination; and apharmaceutically acceptable carrier, vehicle or diluent, wherein saidcomposition comprises antibody in a dose range of 1.25 μg antibody/kgbody weight to 2.5 μg antibody/kg body weight.
 2. The pharmaceuticalcomposition of claim 1, wherein said composition comprises a doseselected from 1.25 μg antibody/kg body weight, 1.3 μg/kg, 1.4 μg/kg, 1.5μg/kg, 1.6 μg/kg, 1.7 μg/kg, 1.8 μg/kg, 1.9 μg/kg, 2.0 μg/kg, 2.1 μg/kg,2.2 μg/kg, 2.3 μg/kg, 2.4 μg/kg, and 2.5 μg antibody/kg body weight. 3.A therapeutically effective pharmaceutical composition comprising: anantibody capable of stimulating remyelination comprising the heavy chainvariable regions CDR1, CDR2 and CDR3 sequences as set out in FIG. 5 andSEQ ID NO:7 and the light chain variable region CDR1, CDR2 and CDR3sequences as set out in FIG. 6 and SEQ ID NO:9; the antibody selectedfrom the group consisting of: a human monoclonal antibody mAb sHIgM22(LYM 22), active fragments thereof, or recombinant antibodies derivedtherefrom; a therapeutic compound, drug, or agent useful inneuroregenerative therapy or remyelination; and a pharmaceuticallyacceptable carrier, vehicle or diluent, wherein said antibody comprisesa single unit dose of antibody that is 500 ng.
 4. The composition of anyof claims 1-3 wherein said antibody comprises a heavy chain variableregion amino acid sequence as set out in FIG. 5 and SEQ ID NO: 7 and alight chain variable region amino acid sequence as set out in FIG. 6 andSEQ ID NO:
 9. 5. The composition of any of claims 1-3 wherein thecompound, drug or agent is a therapeutic compound for the treatment ofmultiple sclerosis.
 6. The composition of claim 5 wherein thetherapeutic compound is an anti-inflammatory agent, a steroid, a betainterferon formulation or copolymer
 1. 7. A method of reducing orinhibiting demyelination of the central nervous system in a mammalhaving a demyelinating disease or demyelinating neurological conditionwhich comprises administering to said mammal the composition of any ofclaims 1-3.
 8. The method of claim 7 wherein the compound, drug or agentin the composition is a therapeutic compound for the treatment ofmultiple sclerosis.
 9. The method of claim 7 wherein said disease orcondition is selected from multiple sclerosis, a viral disease of thecentral nervous system, and acute or chronic spinal cord injury.
 10. Themethod of claim 7 wherein said viral disease of the central nervoussystem is post-infectious encephalomyelitis.
 11. The method of claim 7wherein the method of administration is selected from intravenous,intraperitoneal, intrathecal, subcutaneous, sublingual, intramuscular,rectal, respiratory and nasopharyngeal administration.
 12. The method ofclaim 7, wherein the administering step comprises administration in asingle dose.
 13. The method of claim 7, wherein the administering stepcomprises administration in multiple separated or divided doses.
 14. Amethod of stimulating remyelination of central nervous system axons in amammal which comprises administering to said mammal the composition ofany of claims 1-3.
 15. The method of claim 14 wherein the compound, drugor agent in the composition is a therapeutic compound for the treatmentof multiple sclerosis.
 16. The method of claim 14 wherein the method ofadministration is selected from intravenous, intraperitoneal,intrathecal, subcutaneous, sublingual, intramuscular, rectal,respiratory and nasopharyngeal administration.
 17. The method of claim14, wherein the administering step comprises administration in a singledose.
 18. The method of claim 14, wherein the administering stepcomprises administration in multiple separated or divided doses.